CHAPTER

16
PRINCIPLES OF STAINING

Staining is the process whereby tissue components are made visible in
microscopic sections by direct interaction with a dye or staining solution. A colored
compound is used to produce a contrast between different tissues and cellular
components based on their varying affinities for most dyes and stains, so
morphologic changes are more easily identified, physical characteristics and
structural relationships of tissues and their cells can be evaluated, and the presence
or absence of disease can be established.
Most cells are colorless and transparent, and therefore histological sections
have to be stained in some way to make the cells visible. The same is true of
components of the extracellular matrix. Because different parts of the cell are
biochemically different, they take up specific stains to varying degrees. The
main reason why cells are stained is to enhance contrast and visualization of the
cell or certain cellular components under a microscope. Cells may be stained to
highlight metabolic processes, to differentiate between live and dead cells in a
specimen, to demonstrate the relationship between internal and external
structures of the cells, and to identify different types of cells.
A histologic stain is the purified form of a coloring agent or crude dye that is
generally applied in an aqueous solution. The actual staining process may
involve immersing the sample (before or after fixation and mounting) in dye
solution. Certain parts of cells and tissues that are acidic in character (e.g.
nucleus) have greater affinity for basic dyes, while basic constituents (e.g.
cytoplasm) take more of the acid stains. Individual variation of the tissue
constituents regarding these properties will consequently produce variation in
colors under the microscope. Many dyes, however, require the use of a mordant -
a chemical compound that reacts with the stain to form an insoluble, colored
precipitate on the tissue and make the staining reaction possible. When excess
dye solution is washed away, the mordanted stain remains.
It is important to remember that the colors of stains are not the real color of
a particular tissue, and that a structure that appears as one color using one stain,
may be a quite different color using another stain. The great majority of routine
histology is done with hematoxylin and eosin (H&E) staining, because it is
quick, cheap and informative. It involves the use of two contrasting stains, e.g.,
hematoxylin which stains the nuclear detail, and eosin which brings out the
cytoplasmic detail of the cell and the tissue's architecture.

STAINING OF PARAFFIN SECTIONS
Paraffin wax is poorly permeable to most staining solutions and should therefore
be removed from the section prior to staining. This is usually done by immersing
the paraffin section in a solvent (e.g. xylene) two times, at 1-2 minutes duration
each, for sections up to 10 micron thick. Xylene is not miscible with aqueous
solutions and low graded alcohol, and should therefore be subsequently removed
with absolute alcohol, followed by descending grades of alcohol to prevent
damage and detachment of sections. The alcohol is then finally replaced with
water before actual staining of section is performed. Such procedure is the exact
reverse of impregnation and may be summed up by the phrase "Sections to
Water".
After the section is cut and mounted on the slide, it is drained and dried
thoroughly to ensure that all moisture between the section and slide has
evaporated, and that the section is firmly attached to the slide. If drying is not
complete, the section (or part of it), especially from bone and nervous tissue,
may become detached from the slide during the process of staining, usually after
adding the acid differentiator.
If an alcoholic stain is to be used, there is no more need to replace the alcohol
with water. After deparaffinization with xylene, the section is transferred to
decreasing grades of alcohol, and in such instances, the term "Sections to
Alcohol" is used, and the staining procedure is subsequently done unless the
tissue has been fixed in mercuric chloride solution, in which case, the section is
taken “to water”.
After staining, the section is again dehydrated with increasing grades of alcohol
and cleared with two changes of xylene to prepare the section for mounting,
since most mountants are miscible in xylene. The second change of xylene will
also raise the refractive index of the glass slide, thereby reducing light refraction
during microscopic examination. The stained section may be left in xylene for an
indefinite period of time until it is finally mounted on the slide. The section
should not be allowed to stay in alcohol for a Jong time because many stains are
usually removed by prolonged immersion in alcohol.
Sections may float off the slide during staining if the slides are dirty or greasy, or
if the sections have not been left in the paraffin oven long enough to dry and be
fixed in the slide. Sections must be left in the oven for a minimum of 30 minutes
before they are finally stained to avoid such problems.

 HISTOLOGICAL STAINING
Histological staining is the process whereby the tissue constituents and
general relationship between cell and tissue are demonstrated in sections by
direct interaction with a dye or staining solution, producing coloration of the
active tissue component. Micro-anatomic stains, bacterial stains and specific
tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this
category. Histologists have developed many stains which are suited to particular
purposes, allowing cell structures to be differentiated. It is important to
remember that the colors of stains are not the real color of a particular tissue, and
that a structure that appears as one color using one stain, may be a quite different
color using another stain.

METHODS OF STAINING
Direct Staining:
Direct staining is the process of giving color to the sections by using
aqueous or alcoholic dye solutions. In simple (or direct) staining only one dye is
used, which is washed away after 30–60 seconds, prior to drying and
examination. The molecules that make up basic dyes have a positive charge.
This is important because the cell wall and cytoplasm of bacterial cells have a
negative charge. The positively charged dye is attracted to the negatively
charged cells, enhancing the ability of the stain to stick to and color the cells.
Methylene blue is a classic example of a simple stain. This blue stain will color
all cells blue, making them stand out against the bright background of the light
microscope.

Indirect Staining:
Indirect staining is the process whereby the action of the dye is intensified
by adding another agent or a MORDANT which serves as a link or bridge
between the tissue and the dye, to make the staining reaction possible. By itself,
the dye may stain only weakly, if at all. The mordant combines with a dye to
form a colored "lake", which in turn combines with the tissue to form a "tissue-
mordant-dye-complex" that is rendered insoluble in ordinary aqueous and
alcoholic solvents. This allows subsequent counterstaining and dehydration to be
carried out easily. It is an integral part of the staining reaction itself, without
which no staining could possibly occur. A mordant may be applied to the tissue
before the stain, or it may be included as part of the staining technique, or it may
be added to the dye solution itself. Examples of mordants are potassium alum
with hematoxylin in Ehrlich's hematoxylin, and iron in Weigert's hematoxylin.
 Fig. 16-1. Mordant and Accentuator

An ACCENTUATOR, on the other hand, is not essential to the chemical
union of the tissue and the dye. It does not participate in the staining reaction,
but merely accelerates the reaction. Examples are potassium hydroxide in
Loeffler's methylene blue and phenol in carbol thionine and carbol fuchsin.

PROGRESSIVE STAINING
Progressive staining is the process whereby tissue elements are stained in a
definite sequence, and the staining solution is applied for specific periods of time
or until the desired intensity of coloring of the different tissue elements is
attained. Once the dye is taken up by the tissue, it is not washed or decolorized.
The differentiation or distinction of tissue detail relies solely on the selective
affinity of the dye for different cellular elements.

REGRESSIVE STAINING
With this technique, the tissue is first overstained to obliterate the cellular
details, and the excess stain is removed or decolorized from unwanted parts of
the tissue, until the desired intensity of color is obtained. Routine Hematoxylin
and Eosin (H&E) staining is the most common method utilized for
microanatomical studies of tissues, using the regressive staining which consists
of overstaining the nuclei, followed by removal of superfluous and excessive
color of the tissue constituent by acid differentiation.

DIFFERENTIATION (DECOLORIZATION) is the selective removal of
excess stain from the tissue during regressive staining in order that a specific
substance may be stained distinctly from the surrounding tissues.
A staining procedure that differentiates or distinguishes between types of
bacteria is termed as a differential staining technique. Methods for simple
staining impart same color to all bacteria and other biological material, may
cause slight variation in shade. On the other hand, differential staining methods
impart a distinctive color only to certain types of bacteria. In some techniques,
the stains are applied separately, while in other they are applied as a combined
stain.
Differential Staining uses more than one chemical stain to better
differentiate between various microorganisms or structures/cellular components
of a single organism. This is usually done by washing the section in simple
solution (e.g. water or alcohol), or by the use of acids and oxidizing agents. In
general, if the primary stain used is a basic dye, the differentiation is carried out
by an acid solution, while alkaline medium is used for differentiation after
applying an acidic dye. Alcohol acts as a differentiator for both basic and acidic
dyes, probably by simply dissolving out the excess dye. Differential staining is
also used to detect abnormalities in the proportion of different white blood cells
in the blood. The process or results are called a WBC differential. This test is
useful because many diseases alter the proportion of certain white blood cells.
A mordant can act as a differentiating agent. Mordants such as iron alum can
also oxidize hematoxylin to a soluble, colorless compound, so that the tissue
component becomes decolorized. On the other hand, if a section that has been
stained by a mordant dye is allowed to remain in a differentiating agent such as 1
to 2% alcohol, all the dye will be removed. This is actually done as a preliminary
step in re-staining a faded slide. Differentiation is usually controlled by
following exact times specified for staining, or by examination under the
microscope.
One commonly recognizable use of differential staining is the Gram stain.
Gram staining uses two dyes: Crystal violet and Fuchsin or Safranin (the
counterstain) to differentiate between Gram-positive bacteria (large
Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria.

METACHROMATIC STAINING
Most dyes stain tissues orthochromatically, i.e., in color shades that are
similar to the color of the dye itself. Metachromatic staining technique entails
the use of specific dyes which differentiate particular substances by staining
them with a color that is different from that of the stain itself (metachromasia).
Tissue components combine with these dyes to form a different color from the
surrounding tissue. This is particularly employed for staining cartilage,
connective tissues, epithelial mucins, mast cell granules, and amyloid.
At its simplest, the actual staining process may involve immersing the
sample (before or after fixation and mounting) in dye solution, followed by
rinsing and observation. Many dyes, however, require the use of a mordant: a
chemical compound that reacts with the stain to form an insoluble, colored
precipitate. When excess dye solution is washed away, the mordanted stain
remains. Although methyl violets, of which crystal violet is one, do give
metachromatic staining, they are not considered to be the most effective for the
purpose. The azures or toluidine blue are more effective usually. The exception
is for amyloid, when significant metachromasia is given by amyloid deposits
using crystal or methyl violets.

METALLIC IMPREGNATION
Metallic Impregnation is a process where specific tissue elements are
demonstrated, not by stains, but by colorless solutions of metallic salts which are
thereby reduced by the tissue, producing an opaque, usually black deposit on the
surface of the tissue or bacteria. Specific tissue elements are demonstrated, not
by stains, but by colorless solutions of metallic salts which are thereby reduced
by the tissue, producing an opaque, usually black deposit on the surface of the
tissue or bacteria. Ammoniacal silver, for example, is reduced by argentaffin
cells (e.g. in melanin and intestinal glands), forming black deposits seen under
the microscope.
A metallic impregnating agent is different from a stain in that it is not
absorbed by the tissue, but is held physically on the surface as a precipitate or as
a reduction product in certain tissue components. The most valuable metals for
this purpose are gold (gold chloride) and silver (silver nitrate).
Metallic silver deposits are sometimes adventitiously formed in sections;
hence, all reagents to be used should be chemically pure, glassware should be
clean and a formalin-laden atmosphere which is apt to precipitate such pigment
disposition should be avoided. Also, since ammoniacal silver solutions are
potentially explosive, care should be taken to prepare all solutions in clean
containers just before use, and silvered glassware should be avoided. Flexible
plastic containers may be used instead. Solutions should never be exposed to
sunlight if explosion is to be avoided, and all unused reagents should be
immediately inactivated by sodium chloride or dilute hydrochloric acid solution
and discarded. The use of metallic instruments should be avoided when handling
sections for metallic impregnation.

VITAL STAINING
Vital staining is the selective staining of living cell constituents,
demonstrating cytoplasmic structures by phagocytosis of the dye particle
(cytoplasmic phagocytosis), or by staining of pre-existing cellular components
(true vital staining), as in the staining of mitochondria by Janus green. Vital
stains are excluded by the living cells but taken up by the already dead cells as in
the vital staining of reticulo-endothelial system with trypan blue, or propidium
iodide for eukaryotic cells. The usual purpose is to reveal cytological details that
might otherwise not be apparent; however, staining can also reveal where certain
chemicals or specific chemical reactions are taking place within cells or tissues.
The nucleus of a living cell is resistant to vital stains, and therefore is not
demonstrated. In fact, demonstration of nuclear structures during vital staining
suggests permeability of the membrane of the dye, signifying the death of the
cell.

INTRAVITAL STAINING
Intravital staining of living cells is done by injecting the dye into any part of
the animal body (either intravenous, intraperitoneal or subcutaneous), producing
specific coloration of certain cells, particularly those of the reticulo-endothelial
system. Common dyes used are lithium, carmine and India ink.

SUPRAVITAL STAINING
Supravital staining is a method of staining used in microscopy to examine
living cells that have been removed from an organism. It differs from intravital
staining, which is done by injecting or otherwise introducing the stain into the
body. Those that enter and stain living cells are called supravital stains (e.g. New
Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining). However,
these stains are eventually toxic to the organism, some more so than others.
Partly due to their toxic interaction inside a living cell, when supravital stains
enter a living cell, they might produce a characteristic pattern of staining
different from the staining of an already fixed cell (e.g. "reticulocyte" look
versus diffuse "polychromasia"). To achieve desired effects, the stains are used
in very dilute solutions ranging from 1:5,000 to 1:50,000. Note that many stains
may be used in both living and fixed cells. Thin slices of tissues are placed in
small staining dishes and enough staining solution is added to cover the tissue.
Common dyes used are:
1 Neutral red -probably the best vital dye.
2. Janus green-especially recommended for mitochondria.
3. Trypan blue -one gram of dye is dissolved in 100 ml. of sterile distilled
water to be used immediately; it is dangerous to allow the suspension to
stand for more than one hour, because it is likely to become toxic to the
cell.
4. Nile blue
5. Thionine
6. Toluidine blue

 HEMATOXYLIN AND EOSIN (H & E) Staining

Hematoxylin and Eosin (H&E) staining is the corner stone of tissue-based
diagnosis. The process stains thin tissue sections so that pathologists can
visualize tissue morphology. The process uses a hematoxylin dye to stain cell
nuclei (and other parts) blue and an eosin dye to stain other structures pink or
red. Hematoxylin binds strongly to acids and consequently binds to nuclear DNA
and stains nuclei blue. Properly applied, this technique provides exceptional
detail of tissue structure and the makeup of the cells. This detail is required for
tissue-based diagnosis, particularly in the detection and classification of
infection, cancer or metabolic disease.
Routine H&E staining plays a significant role in tissue-based diagnosis by
coloring otherwise transparent tissue sections, and allowing cell structures
including the cytoplasm, nucleus, and organelles and extra-cellular components
to be clearly visible under the microscope. In a histology laboratory, all
specimens are initially stained with H&E and additional stains are only ordered
if additional information is needed to provide a more detailed analysis.
Staining with H&E is very reliable although it does show some variation
depending on the exact formulation of the stain, and the stain density is
considerably affected by the thickness of the sections – thicker sections take up
more stain. It is also generally done before any additional staining techniques,
because histology with H&E can confirm the basic tissue type and help to
localize the lesion. (The term lesion is used by pathologists to indicate any area
of damage, infection, inflammation, tumor, necrosis or otherwise abnormal
tissue.). Since most cell structures are transparent, very little detail of the
structure can be seen, unless the cells are stained. The same is true of
components of the extracellular matrix. Because different parts of the cell are
biochemically different, they take up specific stains to varying degrees.

ROUTINE H&E STAINING in Paraffin Embedded Section (Regressive
Staining)
Fixation: Most fixatives can be used except osmic acid solutions which inhibit
hematoxylin.
Procedure:
1. Clear paraffin embedded sections in first xylene bath for 3 minutes.
2. Transfer to second xylene bath for 2 to 3 minutes.
3. Immerse in first bath of absolute ethyl alcohol for 2 minutes.
4. Transfer to a bath of 95% ethyl alcohol for 1 or 2 minutes.
 5. Rinse in running water for 1 minute.
6. Stain with Harris alum hematoxylin for 5 minutes (Ehrlich's
hematoxylin requires 15-30 minutes).
7. Wash in running tap water to remove excess stain.
8. Differentiate in 1% acid-alcohol (1 ml concentrated HCl to 99 ml. of
80% ethyl alcohol) for 10-30 sec. monitoring the changes in color
microscopically until only the nuclei are stained.
9. Rinse in tap water.
10. Blue in ammonia water (average of 5 minutes) or 1% aqueous
lithium carbonate until the sections appear blue (about 30 seconds).
11. Wash in running water for 5 minutes.
12. Counterstain with 5% aqueous eosin for 5 minutes. If alcoholic
eosin is used, the time can be reduced to 30 seconds or 1 minute.
13. If aqueous eosin is used, wash and differentiate in tap water under
microscope control until the nuclei appear sharp blue to blue black and
the rest of the tissue appear in shades of pink. If alcoholic solution is
used, differentiate with 70% alcohol.
14. Dehydrate, clear and mount.
NOTE:
For tissues fixed with mercuric chloride, the staining time in hematoxylin
should be increased slightly while duration of eosin staining should be reduced.
The mercury should be removed using a 0.5% solution of iodine in 80 to 95%
alcohol and rinsed in water. The iodine is then removed by placing the slide in
3% sodium thiosulfate solution for 1 to 5 minutes and washing it well in running
water for 3 to 5 minutes. Alternatively, mercury deposits may be removed after
sections are hydrated, by immersing the sections in Gram's or Lugol's iodine for
5 minutes, followed by sodium thiosulfate and subsequently washing the section
in water prior to staining.
Staining may be prolonged for chromium and osmium fixed tissues (e.g.
Flemming's fluid), for tissues subjected to long acid decalcification, and after
prolonged storage in acid formalin or 70% alcohol.

FROZEN SECTION STAINING
Frozen sections mounted on the slides may be stained as in paraffin sections
although the duration of staining is usually shorter. Sections may be mounted in
an aqueous medium directly from water if necessary. Frozen sections may be
stained by picking up sections on albuminized slides and drying them quickly or
by simple direct staining on a wet slide with an eye dropper. The following
staining methods are commonly employed for frozen sections, the choice
depending upon the personal preference of the pathologist and the type of tissue
section to be stained.
1. Hematoxylin-Eosin method
2. Thionine method
3. Polychrome Methylene Blue method
4. Alcoholic Pinacyanol method (used also for supravital staining of
mitochondria and primarily for color sensitization in photography)

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)
1. Orient section in the block and freeze with liquid nitrogen.
2. Cut cryostat sections at 5-10 micron.
3. Mount sections on to albuminized slides and dip in 10% formalin to
fix.
4. Rinse rapidly in water.
5. Stain with Harris hematoxylin for 30-45 seconds.
6. Rinse in tap water.
7. Blue in ammonia water for 5 seconds.
8. Rinse in tap water.
9. Counterstain with 5% aqueous eosin or 1% alcohol eosin for one
minute.
10. Rinse in tap water.
11. Dehydrate in increasing concentrations of alcohol.
12. Clear with xylene.
13. Mount with cover slide.

It is somewhat less favored than regressive staining due to the difficulty of
producing sufficiently intense progressive staining of cell structures without
staining other parts, thereby resulting in diffused color and obscured details. For
convenience, reagents for this rapid H&E stain are generally arranged in
sequence using a series of Coplin jars. This method takes only 5-10 minutes and
produces well-differentiated sections that are semi-permanent and can be stored.
The remaining portion of tissue must be kept for routine processing and are
made for comparison with frozen sections.

 Fig. 16-2. Passing slides through a series of solutions

Precautions in Staining
Stains on the skin should be avoided not only because they are signs of poor
technique but because stains are health hazards per se, being slowly absorbed by
the skin and eventually producing side effects. Stains may be effectively
removed from the skin by prompt topical application of 0.5% acid alcohol,
followed by rinsing with tap water.
Failure of staining may be due to paraffin, fixative, or decalcifying solution
that has not been thoroughly washed out and removed. Early fixation in alcohol
before paraffin embedding may have been incorrect, for which no remedy can be
made. Alternatively, the staining solution may be faulty. Hematoxylin solutions
may not have been properly and sufficiently ripened. Hematoxylin must not be
used too soon after preparation to ensure complete ripening. Impurities found in
the dye or in the water solvent will affect not only the solubility of the dye but
even the intensity of the staining reaction, necessitating purification and filtering
of the dye. Stains that have already been deteriorated should be replaced.
If, after staining, sections are fuzzy and do not appear clear under the
microscope, xylol should be replenished. There may be water in the absolute
alcohol, moisture in the coverslip, or too much egg albumin on the slide, thereby
obliterating the image of the stained tissue. And often, acid-alcohol decolorizer
may not have been completely removed, or a film from alkaline alcohol may
have been carried along. To remedy the condition, the section is placed in a
Coplin jar containing xylol to dissolve the adhesive. The slide is run back thru
the various processes up to the point where the fault was; a fresh solution is
used, and the tissue is re-stained.
Stains may be saved and used again for as long as they have not lost their
staining properties. Sections are usually rinsed with distilled water before
placing them in used stains. Formation of precipitate in staining solution and
poor staining results signify loss of staining property and hence, the stain should
be discarded and replaced with a fresh solution.
Failure of sections to remain on the slide during staining could have been
due to a dirty or oily slide. Slides may have been carried thru the first alcohol
baths too fast, resulting in a rapid but incomplete dehydration; or paraffin
sections may not have been thoroughly spread on the slide when mounted.
Albumin fixative may be too old, as suggested by the loss of its clear color, or by
emission of an odor. To avoid this, adhesives should be prepared in small
amounts (around 1 ounce) which may last for 2-3 months.

COLLODIONIZATION OF SECTIONS
Paraffin ribbons containing air bubbles, torn or inadequately infiltrated
sections are likely to float from the slide when deparaffinized and stained. They
are more firmly attached by coating the slide with dilute (thin) celloidin
solutions, a process known as collodionization, which is also recommended for
sections that will be subjected to strong alkaline or acid solutions and for tissues
that contain glycogen for demonstration.
Procedure:
1. Deparaffinize in xylene.
2. Dehydrate thru absolute alcohol.
3. Dip individual slides in Coplin jar containing dilute ether alcohol
solution.
4. Dip in dilute ether solution of celloidin (thin celloidin).
5. Hold slide on one end for 1/2 to 1 minute to drain or until the section
begins to whiten around the edges.
6. Wipe off the back of the slide and place in 80% alcohol for 3-5 minutes
to harden the celloidin.
7. Stain as desired.
Sections may be transferred from one solution to another with a bent glass
rod (as in frozen sections), but because they are thicker, they may be handled by
means of forceps instead.
Cellulose nitrate (celloidin) is soluble in absolute alcohol, and will be
removed if absolute alcohol is used in the final dehydration prior to clearing of
stained sections. Instead, sections treated with 95% alcohol may be transferred to
a mixture of equal parts of chloroform, absolute alcohol and xylene (C.A.X,)
then treated with xylene and mounted in Xam.

RE-STAINING OF OLD SECTIONS
Old, bleached or faded sections may be re-stained: the slide is usually
immersed in xylene for 24 hours, or gently heated until the mounting medium
begins to bubble. The coverslip may then be removed by lifting it with a
dissecting needle. The section is placed in xylene for up to 24 hours to remove
the remaining balsam and then brought down to water. It is placed in a 0.5
potassium permanganate solution for 5-10 minutes, rinsed in tap water and
subsequently immersed in 5% oxalic acid for 5 minutes or until the section is
decolorized. After washing it again in running tap water for another 5 minutes,
the section may then be re-stained with the appropriate staining technique.

HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
Histochemical staining is the process whereby various constituents of
tissues are studied thru chemical reactions that will permit microscopic
localization of a specific tissue substance. Chemical ions such as calcium,
molecules such as bile pigments, and biopolymers such as cellulose, DNA and
specific enzymes are among the tissue components that can be identified using
histochemical staining techniques. In enzyme histochemistry, the active staining
reagent serves as a substrate upon which the enzymes act, and the final
coloration produced is from the substrate rather than the tissue. In many
instances, histochemical methods used to stain several chemical constituents will
also ultimately stain the tissue itself, thereby producing an overlapping of
techniques. The staining techniques employed for histochemistry are also usually
applied for staining of histologic structures. Examples of such type of stains are
Perl's Prussian blue reaction for hemoglobin, and Periodic Acid Schiff staining
for carbohydrates.
IMMUNOHISTOCHEMICAL (IHC) STAINING is a combination of
immunologic and histochemical techniques using a wide range of polyclonal or
monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and
demonstrate tissue antigens (e.g., proteins) and phenotypic markers under the
microscope. Immunohistochemical staining is widely used in the diagnosis of
abnormal cells such as those found in cancerous tumors, in the localization of
biomarkers and differentially expressed proteins in different parts of a biological
tissue, and in the detection of specific molecular markers that are characteristic
of particular cellular events such as proliferation or cell death (apoptosis).
Visualizing an antibody-antigen interaction can be accomplished in a
number of ways. In most cases, an antibody is conjugated to an enzyme, such as
peroxidase, that can catalyze a color-producing reaction. Alternatively, the
antibody can also be tagged with a fluorophore, such as fluorescein or
rhodamine. Immunohistochemical staining techniques are used to label defined
antigens with monoclonal and polyclonal antibodies. Commercially produced
antibodies most frequently originate from mice, and less frequently from rabbits.
The degree of autolysis or putrefaction, the selection of fixation medium,
fixation duration, incubation period, and concentration of the selected antibodies
can be crucial factors that can affect the results of immunohistochemical staining
protocols. Unlike conventional histological staining methods,
immunohistochemical techniques are based on antigen–antibody bindings, which
can be affected by inappropriate fixative selection and duration. The current
recommendation for immunohistochemical techniques is a maximum of 4%
neutral buffered formaldehyde solution and, for some antibodies, fixation time
can be up to a maximum of 48 h. Microwave-based fixation of tissue in
formaldehyde may have an adverse effect on immunohistochemical staining.

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preformed metal complexes. Histochemie, 35: 361.
McManus JFA, Mowry RW. (1964) Staining Methods. Histologic and Histochemical. Harper & Row,
London.
Pearse AGE. (1980) Histochemistry, Theoretical and Applied, Vol. 2, 4th ed., Churchill Livingstone,
Edinburgh.
Pearse AGE. (1985) Histochemistry, Theoretical and Applied, Vol. 3, 4t11 ed., Churchill Livingstone,
Edinburgh.
Penney DP, Powers JM, Frank M, Churukian C. (2002) Biotech Histochem 77 (5–6): 237–275.
Reynolds JI, Moyes RB, Breakwell DP. (2009) "Differential staining of bacteria: acid fast stain".
Current Protocols in Microbiology. Appendix 3: Appendix 3H.
Ryan KJ, Ray CG. eds. (2004) Sherris Medical Microbiology (4th ed.) McGraw Hill. pp. 232–3.
Sheehan DC, Hrapchak BB. (1980) Theory and practice of histotechnology. 2nd ed. St. Louis, MO: The
C.V. Mosby Company.
Suvarna SK, Layton C, Bancroft JD. (2013) Bancroft’s Theory and Practice of Histological Techniques.
7th ed. Churchill Livingstone, Elsevier.
Wells J. (1988) A Technique for Staining the Superficial Cells of Plucked Hair Follicles and Other Solid
Tissues, Stain Technology, Vol 63, No3.


 CHAPTER 17
STAINS AND STAINING SOLUTIONS

Biological stains or coloring substances are prepared from dyes which may
generally be divided into two categories:
1. Natural dyes - e.g., cochineal dyes, logwood dyes, and vegetable
extracts
2. Synthetic (artificial) dyes - e.g., aniline or coal tar dyes

NATURAL DYES
Natural dyes are those obtained from plants and animals, previously utilized
for dyeing of wool and cotton. Among the most common natural dyes available
are: 1. Hematoxylin
2. Cochineal dyes and its derivatives
3. Orcein
4. Saffron

1. HEMATOXYLIN
Hematoxylin is a natural dye derived by extraction from the core or the
heartwood of a Mexican tree known as "Hematoxylin Campechianum”. It is by
far the most valuable staining reagent used by the cytologist due to its powerful
nuclear and chromatin staining capacity, and its striking polychrome properties
which may be produced with proper differentiation. It may be used after almost
any fixative and is a permanent stain.
Hematoxylin itself is not a true basic dye. The active coloring agent is
hematin, which is formed by the oxidation of hematoxylin, a process known as
"ripening." This is usually accomplished by exposing the substance to air and
sunlight, thereby oxidizing hematoxylin (natural ripening). Such a process is
slow and takes as long as 3-4 months, but it can be accelerated by adding strong
oxidizing agents such as hydrogen peroxide, mercuric oxide, potassium
permanganate, sodium perborate or sodium iodate which converts hematoxylin
to hematin almost instantaneously by chemical oxidation (artificial ripening),
so that the staining solution is ready for use immediately after preparation. It is
essential that the oxidant be used in correct amount, since excessive oxidation
(over-ripening) leads to production of other useless compounds. Using the least
amount of oxidant will result in satisfactory staining and longer life of the stain.
Ripened hematoxylin is seldom used alone due to its inherent low affinity
for the tissue itself. It is most frequently used in combination with alum, iron,
chromium and copper salts, which act as mordants catalyzing or forming links
between the hematin stain and the tissue.
Mordants are substances that combine with the tissue and the staining
solution, forming a "bridge" that allows staining reaction to take place.
Alum hematoxylin stains are recommended for progressive staining of
tissues, and are usually counterstained with Eosin, Congo Red and Safranin.
Both the Ehrlich’s solution and the Harris’ solution contain Alum Hematoxylin.
Rapid ripening of Ehrlich’s reagent, however, is brought about by the addition
of Sodium Iodate; while Harris solution is ripened with Mercuric Chloride.
Iron hematoxylin compounds are used only for differential or regressive
staining, using Acid-Alcohol as a differentiating agent. An example of an Iron
Hematoxylin compound is Weigert’s Stain using Iron (Ferric) Chloride.
Copper hematoxylin solutions are utilized for the study of
spermatogenesis.
Hematoxylin and eosin (H&E) staining protocol is used frequently in
histology to examine thin sections of tissue. Hematoxylin stains cell nuclei
blue, while eosin stains cytoplasm, connective tissue and other extracellular
substances pink or red. Eosin is strongly absorbed by red blood cells, coloring
them bright red.
In a skillfully made H & E preparation the red blood cells are almost
orange, and collagen and cytoplasm (especially muscle) acquire different
shades of pink. When the staining is done by a machine, the subtle differences
in eosinophilia are often lost. Hematoxylin stains the cell nucleus and other
acidic structures (such as RNA-rich portions of the cytoplasm and the matrix of
hyaline cartilage) blue. In contrast, eosin stains the cytoplasm and collagen
pink.

2. COCHINEAL DYES
Cochineal dye is an old histologic dye extracted from the female cochineal
bug (Coccus Cacti), which is treated with alum to produce the dye, carmine. It
is widely used as a powerful chromatin and nuclear stain for fresh material and
smear preparations. When combined with picric acid (picrocarmine), it is
extensively used in neuropathological studies; and when combined with
aluminum chloride (Best's carmine stain), it is used for the demonstration of
glycogen.

3. ORCEIN
Orcein is a vegetable dye extracted from certain lichens which are
normally colorless, but which, when treated with ammonia and exposed to air,
produce blue or violet colors. It is a weak acid, is soluble in alkali, and is
mainly used for staining elastic fibers.
Litmus is also obtained from lichens, treated with lime and soda, and
exposed to ammonia and air. It is, however, not used as a cytological stain
because of its poor staining property. It is instead, used mainly as an indicator.

SYNTHETIC DYES
Synthetic dyes are sometimes known as "Coal Tar Dyes" since they were
originally manufactured from substances that have been taken from coal tar.
They are derived from the hydro-carbon benzene (C6H6), and are collectively
known as Aniline Dyes.
Chromophores are substances with definite atomic groupings and are
capable of producing visible colors. Simple benzene compounds which contain
such substances are known as chromogens. These are different from the dyes in
that any color that they impart to the tissue is not permanent and can, therefore,
be easily removed. Before a chromogen can properly be called a dye, it must
have the property of retaining its color in the tissue. This property is acquired
by the addition of an auxochrome, an auxiliary radical or substance which
imparts to the compound the property of electrolytic dissociation, thereby
altering the shade of the dye, enabling it to form salts with another compound,
and ultimately retaining its color.
A dye, therefore, should consist of a chromophore and an auxochrome
group attached to a hydrocarbon benzene ring. The coloring property is
attributed to the chromophore, and the dyeing property to the salt-forming
auxochrome.
Depending on where the coloring substance (chromophore) is found, dyes
may be classified into three groups:

1. Acid Dyes - where the active coloring substance is found in the acid
component, and the inactive base, e.g. acid fuchsin, is usually the sodium salt
of a sulfonate of rosaniline. One example of such a dye is picric acid, which has
the ability to form salt with an alkali.
Picric acid is outstanding in the sense that it is the only substance so far
that can fix, differentiate and stain tissue all by itself. It may be employed as a
counterstain to basic cytoplasmic stains, to acid fuchsin in Van Gieson's
connective tissue staining, or to crystal violet for the microscopic study of
fungi. It may also be used as a fixative, as a decalcifying agent, or as a tissue
softener.
Trichloracetic acid, picric acid and chromium-fixed tissues usually take in
acidic dyes more readily. Basic cell structures (collagen, eosinophilic granules
of leukocytes, etc.) have an affinity for the acid dye ions and are regarded as
acidophilic.

2. Basic Dyes - where the active coloring substance is found in a basic
component that combines with the acid radical (usually taken from sulfuric,
acetic or hydrochloric acid).
An example of a basic nuclear stain is methylene blue, which may be used
both as an indicator and as a dye. It is very widely used in microbiology for
bacterial staining.
Tissues fixed with mercuric chloride and formaldehyde usually favor
staining with basic dyes. Acidic cell structures (chromatin, mucus, cartilage
matrix etc.) have an affinity for basic dye ions and are therefore regarded as
basophilic.

3. Neutral Dyes - are formed by combining aqueous solutions of acid and
basic dyes, capable of staining cytoplasm and nucleus simultaneously and
differentially. Because they are made up of large molecular complexes, neutral
dyes are insoluble or barely soluble in water, but they are usually soluble in
alcohol. Ethyl alcohol or acetic acid-fixed tissues, on the other hand, readily
take in both basic and acidic dyes.
Examples of neutral dyes are Romanowsky dyes used in hematology,
Giemsa's stain, and Irishman's stain for leukocyte differentiation.

COMMON STAINING SOLUTIONS

HEMATOXYLIN
Hematoxylin is the staining solution most commonly used for routine
histologic studies. The mordants used to demonstrate nuclear end cytoplasmic
structures are alum and iron, forming lakes or colored complexes (dye​-
mordant-tissue complexes), the color of which will depend on the salt used.
Aluminum salt lakes are usually colored blue while ferric salt lakes are colored
blue-black.
The most commonly used staining system is called H&E (Hematoxylin
and Eosin). H&E contains the two dyes hematoxylin and eosin.
Hematoxylin can be considered as a basic dye (general formula for basic
dyes is: Cl- dye). Hematoxylin is actually a dye called hematin (obtained from
the log-wood tree) used in combination with aluminum ions (Al3+). It is used
to stain acidic (or basophilic) structures a purplish blue. (Hematoxylin is not
strictly a basic dye, but it is used with a 'mordant' that makes this stain act as a
basic dye. The mordant (aluminum salts) binds to the tissue, and then
hematoxylin binds to the mordant, forming a tissue-mordant-hematoxylin
linkage).
Eosin is an acidic dye: it is negatively charged (general formula for acidic
dyes is: Na+ dye-). It stains basic (or acidophilic) structures red or pink. This is
also sometimes termed 'eosinophilic'. Thus the cytoplasm is stained pink, by
H&E staining.
Thus the nucleus is stained purple by H&E staining. This means that the
nucleus, and parts of the cytoplasm that contain RNA stain up in one color
(purple), and the rest of the cytoplasm stains up a different color (pink).

Aluminum Hematoxylin Solutions
Aluminum (alum) hematoxylin stains are recommended for progressive
staining of tissues, (i.e. staining for a predetermined time to adequately stain
the nuclei but leave the background tissue relatively unstained, to be later
counterstained with eosin, Congo red or safranin). The alum hematoxylins can
also be used for regressive staining, meaning that the section is overstained,
and then di fferentiated in acid alcohol followed by "blueing".
Aluminum salts give a blue lake, and increase the selectivity for nuclei,
especially if acid is added or is used as a differentiating agent. The two main
alum hematoxylin solutions employed are Ehrlich's hematoxylin and Harris
hematoxylin solutions. Rapid ripening of Ehrlich's reagent is brought about by
the addition of sodium iodate; while Harris solution is ripened with mercuric
chloride.
Alum or potassium aluminum sulfate, when used as the mordant, usually
dissociates in an alkaline solution, combining with -OH of water to form
insoluble aluminum hydroxide. In the presence of excess acid, aluminum
hydroxide cannot be formed, with ultimate failure of aluminum hematoxylin
dye-lake to form, due to lack of -OH ions. Hence, acid solutions of alum
hematoxylin become red. During staining, alum hematoxylin stained sections
are usually passed on to an alkaline solution (e.g. 1% hydroxide) in order to
neutralize the acid and free the OH group, to form an insoluble blue aluminum
hematin-tissue-lake. Such procedure is known as blueing.
For blueing of alum-hematoxylin -stained sections, warm (40° to 50°C) tap
water is commonly used, since it is generally sufficiently alkaline. When tap
water is not sufficiently alkali ne, or is even acid, and is unsatisfactory for
blueing hematoxylin, lithium carbonate (1% w/v in water), bicarbonate (0.2 to
0.5% w/v in tap water), and potassium or sodium acetate may be used.
Alternatively, Scott's Tap Water Substitute (T.W.S.) consisting of 33.5 gm.
NaHC04 and 20 grams MgS04, in 1000 cc of water, with thymol (to inhibit the
formation of molds), is used to accelerate blueing of thin paraffin sections.
Blueing with ammonia, lithium carbonate or Scott's Tap Water Substitute
has more rapid action (about 15, 30 and 60 seconds respectively), compared to
the 5 to 15 minutes required for warm tap water to "blue" hematoxylin.
Ammonia water, used to blue stains, may be prepared by mixing 2 cc. of strong
ammonium hydroxide with 98 cc of tap water. Ammonia (0.5 to 1% in 80%
alcohol) may be "hard" on delicate tissues and may loosen and cause sections
to fall off the slides during staining. Lithium carbonate has a tendency to form
crystalline deposits unless the slides are agitated in it and washed well
afterwards.
The use of very cold water slows down the process while warming
accelerates it. In fact, the use of very cold water (below 10°C) for blueing
sections may even produce pink artifact discolorations on the tissue.

Ehrlich's Hematoxylin
FORMULA:
Hematoxylin 2 gm
Absolute ethyl alcohol 100 ml
Aluminum potassium Sulfate 15 gm approximately
Glycerin 100 ml
Distilled water 100 ml
Glacial acetic acid 10 ml

Dissolve hematoxylin in absolute ethyl alcohol with gentle heat. Dissolve
the potassium alum in distilled water and glycerin with gentle heating and
shake (glycerin is added to slow the oxidation process and prolong the shelf life
of hematoxylin). Mix the two solutions and add glacial acetic acid. Expose to
air and sunlight for several weeks or months in a flask lightly plugged with
cotton, shaking daily. Transfer in a well-stoppered bottle and store in a warm
place.
This naturally ripening alum hematoxylin takes about 2 months to ripen,
but its staining property will last for months or years. Hematoxylin may be
partially oxidized iodate to hasten ripening by addition of 0.3 gm Sodium, but
this will also inevitably shorten the shelf life of the stain. As hematoxylin
solution becomes oxidized, the color of the solution will change from purplish
to deep red, while the pungent odor of acetic acid will be replaced by a pleasant
aroma. Glycerin acts as a stabilizer, retards evaporation of the solution, and
appears to slow down ripening, so that it may be added 4-6 weeks after the
initial preparation. Ehrlich's hematoxylin is generally used for regressive
staining, and differentiated with I % hydrochloric acid in 70% alcohol (acid-
alcohol) until the nucleus is selectively stained. Mucopolysaccharide substances
such as cartilage and cement lines of bones are also stained intensely blue. It is
suitable for tissues that have been subjected to acid decalcification, and is
especially useful for tissues that have been become acidic during prolonged
storage in formalin.
Ehrlich's hematoxylin is not an ideal stain for frozen sections. Staining
time is usually 15-40 minutes.

Harris Hematoxylin
FORMULA:
Hematoxylin 1 gm
Absolute ethyl alcohol 10 ml
Ammonium/Potassium alum 20 gm
Distilled water 190 ml
Mercuric oxide (red) 0.5 gm
Glacial acetic acid 10 ml

Dissolve hematoxylin in absolute ethyl alcohol with gentle heating.
Dissolve ammonium or potassium alum in distilled water on a large (500 ml.
capacity) boiling flask or beaker. Add hematoxylin solution and boil. Add
mercuric oxide and plunge immediately into cold water for rapid cooling. A
large beaker should be used, because the violent liberation of oxygen will cause
the solution to explode from a narrow-mouthed flask.
The solution should assume a dark purple color when ripened by mercuric
oxide. The addition of 4% glacial acetic acid will give a more precise nuclear
staining. The solution is then filtered and transferred into a well-stoppered
bottle.
Harris hematoxylin is a good regressive stain that may either be used
immediately or stored for future use, since it remains stable for a long time
(about 6 months). Since most of the alcohol is evaporated in the process of
boiling, 10 ml. of ethyl alcohol may be added to the final solution, to help
prevent the growth of molds. The precipitate that forms on prolonged storage
should be filtered off before use.
 Harris hematoxylin is widely used for routine nuclear staining, in
exfoliative cytology, and for staining of sex chromosomes. The usual staining
time is 5-20 minutes, depending on the batch and age of stain, the nature of
tissue, and the degree of staining required. Best results are obtained when the
solution is made every 2 or 3 months. The formation of precipitate in the stored
staining solution indicates deterioration in nuclear staining property. The stain
should be filtered before use, and staining time may need to be increased at this
stage.

Cole's Hematoxylin
Cole's hematoxylin is another alum hematoxylin solution recommended for
routine purposes, especially used in sequence with Celestine blue. This alum
hematoxylin is artificially ripened with an alcoholic iodine solution. It is ready
for immediate use, but may need filtering after storage, as with Harris
hematoxylin.
FORMULA:
Hematoxylin 1.5 gm
1% Iodine in 95% Alcohol 50 ml Sat. Aq. Ammonium Alum 700 ml
Distilled Water 250 ml

Dissolve hematoxylin in warm distilled water and mix with iodine. Add
alum solution and boil. Cool and filter before use. Staining time is 10 minutes.

Mayer's Hematoxylin
This is an alum hematoxylin that is chemically ripened with sodium iodate.
Like any alum hematoxylin, it can be used as a regressive stain, but it is also
useful as a progressive stain. It is used as a n uclear counterstain to demonstrate
the presence of cytoplasmic glycogen by special stain. It is also used in
instances when acid-alcohol differentiation might destroy or decolorize the
stained cytoplasmic components like mucopolysaccharides. It is used in
Celestine Blue hemalum method of nuclear staining.
FORMULA:
Hematoxylin 1 gm
Sodium iodate 0.2 gm
Potassium alum 50 gm
Citric acid 1 gm
Chloral hydrate 50 gm
Distilled water 1000 ml
Allow hematoxylin, alum and sodium iodate to dissolve in water
overnight. Add chloral hydrate and citric acid. Boil for 5 minutes and cool. The
addition of sodium iodate immediately ripens the hematoxylin. Citric acid is
usually added after potassium alum has been dissolved (by shaking the
solution); however, the addition of 20 ml. glacial acetic acid seems to give
better nuclear staining and a more stable solution. Chloral hydrate is added to
the final solution as a preservative. One disadvantage of Mayer's hematoxylin
is that it can be stored only for 3 to 6 months at the most.

Iron Hematoxylin Solutions
Iron hematoxylin compounds are used only for differential or regressive
staining, using acid-alcohol as a differentiating agent. Two main iron
hematoxylin solutions are employed for routine work in the laboratory:
Weigert's Solution, using ferric ammonium chloride, and Heidenhain's solution,
using ferric ammonium sulfate (iron alum) as mordants. The dye lake obtained
when ferric salts are used as mordants is an intense blue-black one.
They can be applied to tissues fixed in virtually all fixatives, producing
permanent stains, provided all iron mordants have been wiped out. Tissues that
have been stored in alcohol for years and which would ordinarily fail to stain,
will normally take iron hematoxylin. Tissue structures are stained blackish or
grayish, according to the extent of differentiation, producing minimal eyestrain;
hence, making it useful for photomicrography.
Solutions prepared with correct or optimal amounts of iron salts (0.5 g.
metallic iron for each 1 gram of hematoxylin) are used for dense, regressive
staining (e.g. myelin methods) . The stain becomes more selective for nuclei if
acid or an excess of ferric salt is added. Ferric salts ripen hematoxylin rapidly
and are active oxidizing agents; hence, they do not keep well as a prepared
mixture. In mixtures of hematoxylin and ferric salts, the insoluble lake
gradually precipitates out, so that premixed stains are not very stable.

Regaud's Hematoxylin for Mitochondria:
Among the many methods used to demonstrate mitochondria by light
microscopy, the most permanent and the simplest is Regaud's modification of
iron hematoxylin on sections of material fixed in potassium dichromate and
formalin and subsequently mordanted in dichromate. After staining, the slides
are differentiated to remove the hematoxylin from most cytoplasmic components
other than mitochondria. Unfortunately, the results are not uniform: some cells
will be over-stained and some under-stained. Therefore a number of microscopic
fields should be examined.

Weigert's Hematoxylin Solution
FORMULA:
SOLUTION A:
Hematoxylin 1 gm
Absolute ethyl alcohol ml
SOLUTION B:
30% anhydrous ferric chloride 4 ml
Concentrated hydrochloric acid 1 ml
Distilled water 100 ml

Hematoxylin is dissolved in alcohol with gentle heating, while ferric
chloride, hydrochloric acid and water are mixed in a different container. Both
solutions are stable and may be stored separately for 6 weeks before use. Ferric
chloride is usually added to the staining solution just before use, by mixing
equal parts of the two solutions to produce a deep black mixture. The working
solution will remain active for 1-2 days. It changes color from a deep blue-
black-violet, through violet, purple, brown and yellowish brown within 2 to 3
weeks, as it becomes less and less stable. A solution that has turned brown
should be discarded.
Weigert's solution is the standard iron hematoxylin stain used in the
laboratory, especially for demonstrating muscle fibers and connective tissues. It
is particularly recommended when the preceding stains contain acid (e.g. Van
Gieson stain containing picric acid) which decolorizes nuclei stained with alum
hematoxylin.

Heidenhain's Hematoxylin
It is a popular cytological stain, especially for the study of mitosis. It can
be used after almost any fixative. Chromatin material (nuclear network and
chromosomes) blue black.
FORMULA:
MORDANT DIFFERENTIATOR:
Ferric ammonium sulfate 2.5 gm
Distilled water 100 ml
HEMATOXYLIN STAIN:
Hematoxylin 1.5 gm
95% ethyl alcohol 10 ml
Distilled water 90 ml

Hematoxylin is dissolved in ethyl alcohol and added with water, allowed to
ripen for 4-5 weeks, and stored in tightly stoppered bottles. This iron
hematoxylin uses ferric ammonium sulfate as oxidant/mordant, and the same
solution as the differentiating fluid. The mordant differentiator is used separately
during the process of staining, instead of being added to the solution.
Heidenhain's solution is a cytological stain recommended for regressive
staining of thin sections. After staining, all components are black or dark grey ​-
black. The hematoxylin staining is moved progressively from different tissue
structures at different rates using the iron alum solution. Differentiation can be
more easily controlled if the differentiating iron alum solution is diluted with an
equal volume of distilled water or an alcoholic picric acid solution. It is utilized
for the demonstration of both nuclear and cytoplasmic inclusions such as
chromatin, chromosomes, nucleoli, centrosomes, and mitochondria. Voluntary
muscle striations and myelin are also well stained.

Phosphotongstic Acid Hematoxylin (PTAH)
There are many variants of the original Mallory PTAH technique,
combining hematoxylin with 1% aqueous phosphotungstic acid, which acts as
a mordant. Natural ripening of the tungsten hematoxylin solution is achieved
with light and air, but will take some months to ripen.
FORMULA:
Hematoxylin 1 gm
Phosphotungstic acid 20 gm
Distilled Water 1000 ml

Dissolve the solids in separate portions of distilled water. Add together and
stand in the light to ripen for several weeks. Immediate ripening may be
obtained by adding 50 ml of 0.25% aqueous potassium permanganate after the
two solutions are mixed, so that stain can be used the next day, although peak
staining activity is not reached until after 7 days.
When hematin is used instead of hematoxylin to prepare a staining
solution, the oxidation process is not necessary and the staining solution can be
used immediately, but its staining activity is comparatively short-lived.
The color of the solution ranges from reddish-brown to purple, although
this is not a reliable guide for the study of stained tissues. Nuclei, fibrin,
muscle striations, and myofibrils are colored blue while collagen, bone and
cartilage take an orange-red or brownish red to deep brick-red stain.
Staining is usually progressive, hence, microscopic examination of the
materials every hour is recommended. Ninety-five percent alcohol usually
removes the red component of the stain, so that dehydration and rinsing of
sections should be brief.
Phosphotungstic acid hematoxylin stain usually demonstrates structures in
paraffin as well as celloidin and frozen sections. Staining time is usually 12-24
hours.

EOSIN
Eosin is one of the most valuable stains used for differentially staining
connective tissues and cytoplasm. It is a red general cytoplasmic stain that
combines with hemoglobin to give an orange color. It is an acid dye and the
terms acidophilic, oxyphilic and eosinophilic are often used interchangeably. It
may be used after any fixative and is routinely used in histopathology as a
counterstain to hematoxylin, imparting a pink or red color to cytoplasmic
material, cell membranes, and some extracellular structures. It is commonly
used as a background stain because it gives a pleasing and colorful contrast to
nuclear stains, particularly in chromate and picric acid fixed tissues, and in
acid​ decalcified materials which are strongly stained with eosin.
Yellowish (Eosin Y) -is the most commonly used. It is readily soluble in water,
less in alcohol, available in both aqueous and alcoholic solutions, showing a
green yellow fluorescence especially in alcoholic medium. The aqueous stain is
generally used as a I % solution for 15 seconds to 3 minutes, depending on the
tissue, type of fixative and intensity of color desired. Slightly longer staining
time is required after formalin than after Zenker’s solution.
The other eosin compound is Eosin B (eosin bluish or imperial red); it has a
very faint bluish cast. The two dyes are interchangeable, and the use of one or
the other is more a matter of preference and tradition. Eosin S and Eosin B are
now rarely used.

5% Aq ueous Eosin Y
FORMULA:
Eosin Y 5 gm
Distilled water 100 ml
Dissolve in water by gentle heating. Cool and filter. Thymol
crystals may be added to prevent formation of molds.

Eosin, Stock Alcoholic Solution
FORMULA:
Eosin Y 1 gm
Distilled water 20 ml
95% alcohol 80 ml
 Dissolve Eosin Y in water by gentle heating. Cool and add alcohol.
For use, one part of the stock solution is usually diluted with three parts of
80% alcohol. Addition of 0.5 ml. glacial acetic acid for every 100 ml. of stain
will usually give a deeper red stain to the tissue. Differentiation of the eosin
stain ing occurs in the subsequent tap water wash, and a little further
differentiation occurs through the alcohols.
Combining eosin Y and phloxine B produces a cytoplasmic stain that
demonstrates various tissue components more dramatically.

Eosin-Phloxine B Solution
FORMULA:
1% phloxine 10 ml
1% eosin Y 100 ml
95% alcohol 780 ml
Glacial Acetic Acid 4 ml

Romanowsky Stains
The Romanowsky stains are all based on a combination of eosinate
(chemically reduced eosin) and methylene blue (sometimes with its oxidation
products azure A and azure B). Common variants include Wright's stain, Jenner's
stain, Leishman stain and Giemsa stain. All are used to examine blood or bone
marrow samples. They are preferred over H&E for inspection of blood cells
because different types of leukocytes (white blood cells) can be readily
distinguished. All are also suited to examination of blood to detect blood-borne
parasites like malaria.

OTHER STAINS

Acid Fuchsin-Picric Acid (Van Gieson’s Stain) is a mixture of picric acid and
acid fuchsin for demonstration of connective tissues.

FORMULA:
Picric acid, saturated aqueous solution 100 ml
Acid fuchsin (1 % aqueous solution) 5 ml The solution
weakens after long standing and may be strengthened by adding
a few drops of fresh acid fuchsin.

Acid Fuchsin (Masson Stain) may be used to stain collagen, smooth muscle, or
mitochondria. Acid fuchsine is used as the nuclear and cytoplasmic stain in
Mallory's trichrome method. Acid fuchsine stains cytoplasm in some variants of
Masson's trichrome. In Van Gieson's picro-fuchsin, acid fuchsin imparts its red
color to collagen fibers. Acid fuchsin is also a traditional stain for mitochondria.
FORMULA:
Acid fuchsin 1 gm
Glacial acetic acid 1 ml
Distilled water to make 100 ml

Picro-Fuchsin Solution
FORMULA:
1% Acid fuchsin 13 ml
Saturated aqueous picric acid 87 ml

ACRIDINE ORANGE is a basic acridine fluorochrome which permits
discrimination between dead and living cells, giving green fluorescence for
DNA and a red fluorescence for RNA. It is a nucleic acid selective fluorescent
cationic dye useful for cell cycle determination. When bound to DNA, it is very
similar spectrally to fluorescein. Like fluorescein, it is also useful as a non-
specific stain for backlighting conventionally stained cells on the surface of a
solid sample of tissue (fluorescence backlighted staining).

ACRIDINE RED 3B is used to demonstrate deposits of calcium salts and
possible sites of phosphatase activities.

ALCIAN BLUE - is a complex, water-soluble phthalocyanin dye, similar to
chlorophyll, which stains acid mucopolysaccharides by forming salt linkages
with them. It is an excellent stain because it is simple, it produces a striking
blue color, and it is resistant to various counterstaining procedures. It is more
specific for connective tissue and epithelial mucin due to its use as an acid
solution. Alcian blue is often combined with PAS, as it stains acidic mucins
blue, whereas PAS stains neutral mucins red, hence it can be used to distinguish
elements of the extracellular matrix.
FORMULA:
Alcian blue 1 gm
Glacial acetic acid 1 ml
Distilled water add up to 100 ml

ALIZARIN RED S forms an orange-red lake with calcium at a pH of 4.2. It
works best with small amounts of calcium (such as in Michaelis-Gutman
bodies). The alizarin method is also used on the Dupont ACA analyzer to
measure serum calcium photometrically.

ANILINE BLUE is a cytoplasmic stain used for counterstaining of epithelial
sections.
FORMULA:
Aniline blue 1 gm
Distilled water 97.5 ml
Glacial acetic acid 2.5 ml

AZOCARMINE: Nuclei are deep red; cytoplasm is a pale red.

BASIC FUCHSIN - is a plasma stain utilized also for deep staining of acid-
fast organisms, for mitochondria, for differentiation of smooth muscles with the
use of picric acid. It is a main constituent of Feulgen's and Schiff's reagent for
the detection of aldehydes, of Van Gieson's solution for connective tissues,
mucin, and for elastic tissue staining.

a. CARBOL-FUCHSIN
FORMULA:
Basic fuchsi n 1 gm Phenol crystals 5 gm
Absolute ethyl alcohol 10 ml
Distilled water 100 ml
Grind basic fuchsin and phenol together in a mortar. Add
alcohol and then water. Boil in a beaker and stand for 24 hours
to cool. Filter before use.



2. COLEMAN'S FEULGEN REAGENT
FORMULA:
Basic fuchsin 1 gm
Sodium metabisulphite 1 gm
1N hydrochloric acid 10 ml
Distilled water 200 ml
Boil water, remove from heat and add basic fuchsin. Cool and
add sodium metabisulphite and hydrochloric acid. Stand for 24
hours and filter through activated charcoal to get a colorless
filtrate. Store in a refrigerator.

c. SCHIFF'S REAGENT
FORMULA:
Basic fuchsin 1 gm
Sodium metabisulfite anhydrous 1 gm
Distilled water 200 ml
Normal hydrochloric acid 20 ml
Boil water. Add basic fuchsin and dissolve by stirring. Cool to
50°C, filter and add hydrochloric acid. Cool to 25°C and add
sodium metabisulphite. Let stand for 24 hours until the solution
becomes pale-straw in color. Filter through activated charcoal
to form a colorless filtrate. Store in a refrigerator.

d. MALLORY'S FUCHSIN STAIN
FORMULA:
Basic fuchsin 0.5 gm
95% ethyl alcohol 50 ml
Distilled water 50 ml
Dissolve fuchsin in alcohol by gentle heating. Add water, cool,
and filter.

e. ALDEHYDE FUCHSIN (GOMORl'S STAIN)
FORMULA:
Concentrated hydrochloric acid 1 ml
Paraldehyde 1 ml
0.5% basic fuchsin in 70% alcohol 100 ml
Stand at room temperature for 24 hours until the mixture
becomes deep purple in color. Store in refrigerator.

BENZIDINE is used for staining hemoglobin.
FORMULA:
Solution A
Benzidine 0.5 gm
Absolute alcohol 50 ml
Sodium nitroprusside 0.1 gm
Distilled water up to 100 ml
Dissolve benzidine in alcohol. Dissolve sodium nitroprusside in 10
ml. distilled water. Mix and make up to 100 ml. with the remaining
distilled water.
 Solution B
Absolute alcohol 50 ml
Glacial acetic acid 2 ml
Hydrogen peroxide 30% 0.5 ml
Sodium nitroprusside 0. 1 gm
Distilled water up to 100 ml
Both solutions A and B should be freshly prepared.

BISMARCK BROWN - is used as a contrast stain for Gram's technique, in
acid fast and Papanicolau method, and for staining diphtheria organisms.

CARMINE - is used as a chromatin stain for fresh materials in smear
preparations. It is slightly soluble in water at a neutral reaction, and usually
kept in ammoniacal solution which changes its properties due to oxidation. The
most important component of carmine is carminic acid, which is also useful in
industry and analytical chemistry. It can be used for determining the presence
of certain metal ions, such as aluminum. A dry powder is often prepared in the
form of carmine aluminum calcium lake ("carmine alum lake"). It is usually
combined with aluminum chloride to stain glycogen (Best Carmine solution).

Best Carmine Stain (Stock Solution)
FORMULA:
Carmine 2 gm
Potassium carbonate 1 gm
Potassium chloride 5 gm
Distilled water 60 ml
Concentrated ammonia 20 ml
Grind carmine in a mortar and add potassium carbonate and potassium
chloride to water. Boil gently in a large flask (to avoid frothing) for 5
minutes. Cool and add ammonia. Store in a dark bottle inside the
refrigerator. Stain will keep well for about 3 months after preparation.

Best Carmine Working Solution
FORMULA:
Best carmine (stock solution) 2 parts
Ammonia concentrated 2 parts
Absolute methyl alcohol 3 parts

Best's Differentiator
 FORMULA:
Absolute methyl alcohol 40 ml
Absolute ethyl alcohol 80 ml
Distilled water 100 ml

CARMALUM (MAYER'S) SOLUTION -is a mordanted dye acting as a
basic dye and staining acidic substances.
FORMULA:
Carminic acid 0.5 gm
Potassium alum 5 gm
Distilled water 100 ml
Salicylic acid 0.05 gm
Sodium salicylate 0.25 gm
Warm the solution to dissolve the constituents. Filter when cool,
then add salicylic acid and sodium salicylate.

CELESTINE BLUE - Celestine Blue is an oxazine dye used as an alternative
to iron hematoxylin nuclear stain, producing a strong and precise nuclear stain
that is resistant to decolorization by succeeding acid stains and solutions.
Celestine blue forms a strong staining lake with iron alum, acting as a mordant
to bind hematoxylin. It is resistant to strong acid dyes, and is recommended for
routine staining of fixed sections, giving a good nuclear definition when used
in conjunction with alum hematoxylin.
FORMULA:
Ferric ammonium sulfate 5 gm
Distilled water 100 ml
Celestine blue 0.5 gm
Glycerin 14 ml
Dissolve ferric ammonium sulfate in water overnight at room
temperature. Add Celestine blue; Boil for 3 minutes. Cool and filter,
then add glycerin.

CONGO RED - is best known as an indicator, but may be utilized as a stain for
axis cylinders in embryos. It is used as a 4% aqueous solution in staining elastic
tissues and myelin. Congo red is used to identify deposits of protein in tissue
called amyloid.
CRESYL VIOLET - is commonly used in histology to stain nervous tissues.
Cresyl violet stains the acidic components of the neuronal cytoplasm
(specifically Nissl bodies) a violet color.

CRYSTAL VIOLET - is a nuclear or chromatin stain used for staining
amyloid in frozen sections and platelets in blood. Gentian violet is the staining
solution formed by the mixture of crystal violet, methyl violet and dexterin.

ETHIDIUM BROMIDE intercalates and stains DNA, providing a fluorescent
red-orange stain. Although it will not stain healthy cells, it can be used to
identify cells that are in the final stages of apoptosis –such cells have much more
permeable membranes. Consequently, ethidium bromide is often used as a
marker for apoptosis in cells populations and to locate bands of DNA in gel
electrophoresis. The stain may also be used in conjunction with acridine orange
(AO) in viable cell counting. This EB/AO combined stain causes live cells to
fluoresce green whilst apoptotic cells retain the distinctive red-orange
fluorescence.

GIEMSA STAIN – consists of a mixture of methylene-blue and eosin, and it is
used for staining blood to differentiate leukocytes. It is mostly used on methanol-
fixed blood films, where it stains erythrocytes pink and the different types of
leukocyte, allowing their identification according to size and shape of their
nuclei. It also binds to some pathogens, including spirochetes (syphilis),
trypanosomes (sleeping sickness and Chagas disease) and plasmodium (malarial
parasites). In addition it can also be used to stain some bacteria in tissue sections
pink, and it is therefore particularly useful if infection is suspected.
FORMULA:
Giemsa stain 1 gm
Glycerin 66 ml
Absolute methyl alcohol 66 ml
Mix glycerin and Giemsa stain and place in oven at 60°C for 30
minutes to 2 hours then add methyl alcohol.

GOLD SUBLIMATE - is the stain used for metallic impregnation, made up of
gold chloride and mercuric chloride.
FORMULA:
Mercuric chloride 0.4 gm
Distilled water 60 ml
1% Gold chloride (brown) 10 ml
Dissolve mercuric chloride in water by gentle heating. Cool and add
gold chloride, store in a dark place, or mix immediately before use.

IODINE - is probably the oldest of all stains, originally used for microscopic
study of starch granules. It stains amyloid, cellulose, starch, carotenes and
glycogen. It is widely used for removal of mercuric fixative artefact pigments,
and as a reagent to alter crystal and methyl violet so that they may be retained
by certain bacteria and fungi. It may also be used in the form of aqueous or
alcoholic solutions.

Gram's Iodine - is used to identify and differentiate bacteria. For example,
staphylococci, streptococci and pneumococci are gram-positive and stain a deep
blue, whereas coliforms and Neisseria are gram-negative and stain pink.
FORMULA:
Iodine 1 gm.
Potassium iodide 2 gm.
Distilled water 300 ml.
Dissolve potassium iodide in a little water. Add iodine and dissolve
in the remaining water.
Gram's Iodine is used in Gram Weigert method of staining
microorganisms and fibrin in tissue sections.

Lugol's solution or Lugol's iodine
Lugol’s solution is a brown solution that turns black in the presence of starches
and can be used as a cell stain, making the cell nuclei more visible. Iodine is also
used as a mordant in Gram's staining, it enhances dye to enter through the pore
present in the cell wall/membrane.
FORMULA:
Iodine 1 gm
Potassium iodide 1 gm
Distilled water 100 ml
Lugol's Iodine is used as a test for glycogen, amyloid, and corpora
amylacea.

JANUS GREEN B - is used for demonstrating mitochondria during intravital
staining.

MALACHITE GREEN - has sometimes been used for staining erythrocytes is
a weakly basic dye used as a contrast stain for staining ascaris eggs and
erythrocytes, and as a bacterial spore stain; it is also used both as a decolorizer
and as a counterstain. By itself, this dye is not all that good for general
microscopy, being perhaps in the same category as tartrazine. Like tartrazine,
malachite green is primarily a counterstain; this means it gives general color to
areas that have either failed to take up some other, more specific stain or which
have been subjected to de-staining.
FORMULA:
2% aqueous malachite green 220 ml
Glacial acetic acid 30 ml
Glycerol, C.M.
Mix and store in a colored bottle.

MASSON’S TRICHROME is (as the name implies) a three-color staining
protocol. The recipe has evolved from Masson's original technique for different
specific applications, but all are well-suited to distinguish cells from
surrounding connective tissue. Most recipes produce red keratin and muscle
fibers, blue or green staining of collagen and bone, light red or pink staining of
cytoplasm, and black cell nuclei.

METHYL GREEN - stains chromatin green in the presence of an acid. It gives
false positive reactions with certain secretions such as mucin. Methyl green is
used commonly with bright-field microscopes to dye the chromatin of cells so
that they are more easily viewed.

METHYLENE BLUE - is a common basic nuclear stain employed with eosin
to provide marked differentiation of various structures in the tissue. It usually
contains some azures or methylene violet. Methylene blue stains acidic cell
parts (like the nucleus) blue and is a good counterstain with Eosin Y. It can be
substituted for Janus Green B stain or Carmine stain. This methylene blue stain
is a 1% aqueous solution.
"Polychroming" involves the oxidation of methylene blue, resulting in
loss of methyl groups and leaving lower homologues of the dye (azures) and
deaminized oxidation products (thiazoles). The resulting mixture of methylene
blue, azures and thiazoles is known as polychrome methylene blue. It stains
nuclei blue while cartilage matrix, mucin, mast cell granules and connective
tissues generally take a reddish-violet color.
It is a valuable stain for plasma cells and may also be employed in
cytological examinations of fresh sputum for malignant cells, as a bacterial
stain for evaluation and differentiation of bacterial organisms, for diagnosis of
diphtheria, and for vital staining of the nervous tissue.
FORMULA:
Methylene blue 1 gm
 Potassium carbonate 1 gm
Distilled water 100 ml
Mix in a flask that has been lightly plugged with the cotton or
gauze and let stand at 37°C for weeks to oxidize. For use, dilute in
1:5 or 1:10 dilution with distilled water.

This solution is available commercially. For rapid diagnosis, frozen
sections are stained with polychrome methylene blue for I/2 to 1 minute, rinsed
and mounted in an aqueous mountant, blotted dry, or cleared in xylene and
mounted in Clarite, Permount or H.S.R.

Mallory's Phloxine Methylene Blue Stain - originally known as Eosin-
Methylene Blue (EMB) method, this technique produces a sharp nuclear stain
and reveals with marked differentiation the various structures in the tissues,
which should be fixed in Zenker's fluid.

METHYLENE VIOLET - is a metachromatic dye formed whenever
methylene blue is heated in fixed alkali or alkali carbonate, coloring nuclei of
leukocytes reddish-purple in the presence of methylene blue.

NILE RED (also known as Nile blue oxazone) is formed by boiling Nile blue
with sulfuric acid. This produces a mix of Nile red and Nile blue. Nile red is a
lipophilic stain; it will accumulate in lipid globules inside cells, staining them
red. Nile red can be used with living cells. It fluoresces strongly when
partitioned into lipids, but practically not at all in aqueous solution.

OIL RED O is a dye that is more soluble in fat than in water or alcohols, hence
it is used as a stain for neutral lipids. For example when myelin is broken down
in the CNS, in diseases such as multiple sclerosis, macrophages take up the
lipid-rich debris and stain strongly with this dye. The oil red O stain can
identify neutral lipids and fatty acids in smears and tissues. Fresh smears or
cryostat sections of tissue are necessary because fixatives containing alcohols,
or routine tissue processing with clearing, will remove lipids. The ORO is a
rapid and simple stain. It can be useful in identifying fat emboli in lung tissue
or clot sections of peripheral blood.

ORCEIN --is an excellent stain for elastic fibers, and is especially
recommended in dermatological studies due to its ability to demonstrate the
finest and most delicate fibers in the skin.

OSMIUM TETROXIDE
Osmic Acid or Osmium Tetroxide (OsO4) is a selective stain for
unsaturated lipids and for lipoproteins such as myelin, which it stains black.
Osmic acid, aside from being used as a fixative especially for electron
microscopy, may be used to stain fat, although other substances are also stained
simultaneously, thereby preventing specific staining of lipids to be done. Fat,
which reduces osmium tetroxide to osmium dioxide, is stained black, and may
be demonstrated from the tissue by using chrome-osmium solutions or by the
frozen section method.

PERIODIC ACID SCHIFF (PAS) is an all-around useful stain for many
things. It stains glycogen, mucin, mucoprotein, glycoprotein, basement
membranes, capsules, and blood vessels as well as fungi and intracellular
carbohydrates such as glycogen in hepatocytes. Cells that secrete mucus are
also strongly stained. A pre-digestion step with amylase will remove staining
for glycogen. This method depends on the selective oxidation by periodic acid
of free hydroxyl groups on two adjacent hydroxyl groups converting the
alcohols to aldehydes. The aldehydes are then detected by the Schiff reagent,
which stains them reddish purple. Other tissue components stain according to
the counterstain used. Lead-hematoxylin or another basic stain is often the
counterstain.

PHOSPHOTUNGSTIC ACID - is a common negative stain for viruses,
nerves, polysaccharides, and other biological tissue materials. This is an ideal
stain for the demonstration of striated muscle fibers and mitochondria, which
stain blue. A counterstain is often not used.

PICRIC ACID - is employed as a contrast stain to acid fuchsin, for the
demonstration of connective tissue (Van Gieson's stain), as a cytoplasmic stain
in contrast to basic dyes, as a counterstain to crystal violet, as a tissue fixative,
and as a decalcifying agent.

PRUSSIAN BLUE - is an insoluble colored salt of ferric ferrocyanide (an iron
cyanide compound) normally utilized for the manufacture of paints, but may be
used for microanatomical color contrast of specimens and for demonstration of
the blood and lymph vessels by injection (intravital staining).

RHODAMINE B - is used with osmic acid to fix and stain blood and
glandular tissues.

SAFRANIN (or Safranin O) - is a nuclear stain. It produces red nuclei, and is
used primarily as a counterstain. Safranin may also be used to give a yellow
color to collagen.

SILVER NITRATE - is used in 10% aqueous solution to prepare various
dilutions to be used in identification of spirochetes, reticulum and other fiber
stains.

TOLUIDINE BLUE - is a nuclear stain for fixed tissues, used as a substitute
for thionine in fresh frozen tissue sections. It is recommended for staining of
Nissl granules or chromophilic bodies. It is a particularly versatile dye that stains
nuclei blue, and can be used to differentiate different types of granules (e.g.
within mast cells). Because it can permeate the resins that are used to embed
sections for electron microscopy, it is often used as a preliminary stain, to
identify sections that will later be examined by electron microscopy.

VAN GIESON STAIN - binds to collagen in the extracellular matrix, staining it
pink. Often it is combined with a stain for elastic fibers (elastic van Gieson)
which stain black, allowing the two major elements of connective tissue to be
differentiated.

VICTORIA BLUE - is used for demonstration of neuroglia in frozen sections.

VON KOSSA STAIN - is a silver reduction method that demonstrates
phosphates and carbonates, but these are usually present along with calcium.
This stain is most useful when large amounts are present, as in bone.
WRIGHT STAIN - causes blood cells to exhibit four major staining properties
that allow the cell types to be distinguished. Basophilia (affinity for methylene
blue), azurophilia (affinity for the oxidation products of methylene blue called
azures, which are reddish purple), acidophilia (affinity for eosin), and
neutrophilia (affinity for a complex of dyes in the mixture, which are pale lilac).
In a stained blood smear, erythrocytes bind eosin and appear orange to pink,
nuclei purplish blue, basophilic granules very dark bluish purple, eosinophilic
granules red to red-orange, neutrophilic granules reddish-brown to lilac, platelets
violet to purple, and lymphocyte cytoplasm stains pale blue.

OIL SOLUBLE DYES (LYSOCHROMES)
 Lysochromes (oil soluble dyes) are not real dyes in the usual sense of the
word because they do not have auxochrome groups. They give color to lipids
simply because they are more soluble in lipid medium of the tissues, than in
their medium of 70% alcohol. Oil soluble dyes are available in the form of
Sudan Black B. Sudan III and Sudan IV (Scharlach R), used for the
demonstration of intracellular fats which are colored black, orange, and red,
respectively.
In order to penetrate fats, the oil soluble dyes (Sudan dyes) must be
dissolved in organic sol vent, although the solvent vehicle (usually ethanol,
isopropanol or propylene glycol) should be sufficiently dilute (aqueous) to
avoid extracting the lipids themselves.

Sudan Black
Sudan Black is a stain that colors fat droplets black and is the most
sensitive of the oil soluble dyes. It possesses two secondary amino groups per
molecule, making it a slightly basic dye which may cause non-specific staining.
Because of this molecular structure, Sudan black has a much greater affinity for
phospholipids than other lysochromes -coloring neutral lipids (triglycerides) by
simple dissolution of the dye. It has the added advantage of being a more
sensitive coloring agent. The ability of fats to adsorb Sudan Black is related to
dye concentration, temperature and physical state of the fats. Maximal dye
uptake occurs when fat reaches its melting point, so that lipids that are liquid or
semi-liquid at staining temperature will be stained while those that are
crystalline or solid will not be affected by the dye. Sudan Black is prepared as a
0.5% solution boiled in 70% ethanol for 10 minutes under a reflux condenser,
and filtered before use. It is a very unstable solution and should be discarded if
the usual blue-black color turns brownish black. It usually imparts a black color
on intracellular lipids, and is recommended for paraffin sections especially for
tissues fixed in formol calcium with post chroming, demonstrating lipids that
are resistant to paraffin embedding.
Unlike the other Sudan dyes, Sudan Black B stains phospholipids as well
as neutral fats. Sudan black B does not stain crystalline cholesterol, and free
fatty acids tend to be soluble in the ethanolic dye bath.

Sudan IV
Sudan IV (Scharlach R) is different from Sudan Black because it has no
secondary amino group and it does not color phospholipids or the fine lipid
droplets. It is prepared by saturating the dye (Scharlach R) in one part of 2%
benzoic acid (in 70% alcohol), and one part of acetone, forming a very stable
solution which may be used repeatedly as long as it is filtered. Addition of
benzoic acid intensifies fat and prevents rapid deterioration of the solutions. It
is recommended for staining triglycerides (neutral lipids), giving them a deep
and intense red stain.

Sudan Ill
Sudan III was the first Sudan dye to be introduced into histochemistry. It is
also fat soluble, and is good as a fat stain for central nervous system tissues,
giving a less deep and lighter orange stain compared to the darker staining
Sudan IV.

CHIEF SOLVENTS USED FOR STAINS

1. WATER - should always be distilled unless otherwise stated.
2. ALCOHOL - Ethyl alcohol may be used in various concentrations.
Methyl alcohol, if to be used, is usually absolute, and is indicated
especially in the preparation of blood stains, for which reason, it should
be acetone free.
3. ANILINE WATER -Ten ml. of aniline is added to every 1/2 to 1 liter
of hot distilled water, shaken, cooled, and filtered.
4. PHENOL - is used in aqueous solution of 0.5 - 5%.

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CHAPTER 18
STAINING OF CARBOHYDRATES

Carbohydrates are the main sources of energy in the body, mobilized in the
form of monosaccharides (glucose) and stored in the form of polysaccharides,
either in pure form (glycogen), or bound to other substances (mucin). Glycogen
is made up of polysaccharides of glucose, and is normally stored in the liver,
heart and skeletal muscle, but it may be abnormally present in certain diseases.
Mucin is made up of hexosamines (neutral mucopolysaccharides) or mucus that
is secreted by the goblet cells of intestinal mucosa, respiratory lining cells and
certain glands, or found in intercellular substances and connective tissue fibers.
Both glycogen and mucin are stained by the Periodic Acid-Schiff (PAS)
technique.

Periodic Acid Schiff (PAS) Reaction

Periodic Acid Schiff is a histochemical stain that will demonstrate
carbohydrates and other substances in the tissue. The PAS technique uses
periodic acid to specifically oxidize the 1,2 glycol group of polysaccharides and
mucin, liberating aldehydes that are required for the coloration of Schiff's
reagent, thereby producing a red magenta or purplish-pink color.
The treatment with periodic acid oxidizes some of the carbohydrates in the
tissue to aldehydes. The carbohydrates involved are 1-2 glycols. Since not all
carbohydrates include this structure, the PAS is not a method for carbohydrates
in general but only for those which contain 1-2 glycols or closely related
structures, including polysaccharides, mucopoly- saccharides, glycoproteins and
glycolipids. The essential point is that treatment must be able to produce an
aldehyde on the carbohydrate component.
As a general rule, the intensity of PAS reaction is proportional to the content
of sugars (glucose, galactose, mannose, methylpentose, fucose and hexosamines)
present in the reacting substance. In order to have a PAS-positive staining
reaction, oxidation must occur to produce aldehyde. Oxidation may continue
beyond the aldehyde stage, and in such cases, the result will be negative since
this PAS reaction is specific for glycol or glycol amino group where oxidation
does not proceed beyond the aldehyde stage.
 Periodic acid is not the only oxidizing agent that has been recommended for
the oxidation of carbohydrates although it is certainly the most used and
arguably the most effective. Periodic acid has been less popular for fungal cell
walls as staining of carbohydrates in the background may diminish the contrast
between the background pink and the deeper pink of the fungal cell wall. To
overcome this, the oxidant used for fungi is often chromic acid (chromium
trioxide in water) which is different from periodic acid in that it continues to
oxidize the aldehydes it produces. Over oxidation will eventually lead to a pale
or false negative reaction. Some other oxidizing agents have been used as well
including permanganic acid, performic acid, peracetic acid and lead tetracetate.
Although valuable for specific purposes, these are not in common use.
Periodic acid is generally applied to the sections as a 0.5 to 1.0% aqueous
solution for 2 to 20 (average 5) minutes at room temperature. Oxidation beyond
10 minutes increases basophilic methylene blue staining-probably due to
acidification of sulfhydryl groups into sulfonic acid. This can be manifested by
increased affinity of the nuclei for hematoxylin counterstain. A temperature
above 25°C which markedly accelerates the reaction, causing oxidation not only
of aldehydes but also of other groups, e.g., sulfhydryl and disulfide. Solution
must be discarded if it turns brown in color.
Most fixatives can be used with this staining technique, except those that
contain osmic acid, chromates and permanganates. Routine fixation and
decalcification of bone will cause considerable loss of PAS positivity. PAS stain
can be used to demonstrate the following substances: (1) Polysaccharides: These
macromolecules are composed of monosaccharide units joined by covalent
bonds. The main polysaccharide identified through histology staining is
glycogen, which is present in numerous tissues, including skeletal muscle,
cardiac muscle, liver, and kidney.
(2) Neutral mucus substances: PAS is also commonly used to stain and
identify glycoproteins, glycolipids, and neutral mucins, which are produced by
epithelial cells in different organs.
(3) Tissue basement membranes: These PAS-positive thin layers of reticular
connective tissue anchor and support epithelium and endothelium to underlying
connective tissue.
(4) Fungal organisms: The cell walls of some living fungal organisms
contain high levels of carbohydrate, and also stain positive with PAS.

General Principles of the PAS Stain
The reactivity of the PAS technique is based on the structure of the
monosaccharide units. The stain involves periodic acid acting as an agent to
oxidize the carbon-to-carbon bonds between two adjacent hydroxyl groups.
This produces aldehyde groups in the tissue section that then reacts with Schiff
reagent (made up of a mixture of basic fuchsin, hydrochloric acid, and sodium
metabisulphite). The basic fuchsin in the mixture reacts with newly formed
aldehyde groups in the tissue and produces a bright magenta color when the
section is rinsed in water. The intensity of the color is proportional to the
concentration of hydroxyl groups originally present in the monosaccharide
units.
Hematoxylin is typically used as a counter stain to visualize other tissue
elements. However, when PAS is used to demonstrate fungal organisms, a light
green counter stain is preferred.
Diastase (alpha-amylase) digestion may also be used to assist in the
diagnosis of glycogen storage diseases. Diastase hydrolyses and extracts starch,
glycogen, and breakdown products of tissue polysaccharides. When compared
to a slide of tissue containing glycogen, a diastase extraction slide will have no
visible PAS stain.
After examining routine hematoxylin and eosin-stained sections, the
pathologist can order a PAS stain to help with the diagnosis of:
Glycogen storage diseases: These are conditions in which excessive
quantities of glycogen are stored in the liver, muscles, or kidney. PAS is
often routinely used in the clinic to demonstrate glycogen accumulation
in biopsies of these tissues.
Tumors: Glycogen granules can also be present in some tumors,
including some of those that arise in tissues such as the pancreas, lung,
and bladder.
Fungal infection: PAS can be used to visualize some fungal organisms
in tissue sections.
Basement membranes: PAS can be used to highlight abnormal basement
membrane abnormalities – such as in glomerular diseases in the kidney.

Schiff Reagent

The essential component of Schiff reagent is basic fuchsin, which is a
mixture of three dyes (rosanilin, pararosanilin and magenta II). Sulfur dioxide
converts the magenta-colored basic fuchsin into colorless leukofuchsin.
Reoxidation by slow exposure to light and air or by addition of periodic acid
will restore the colorless leukofuchsin to the magenta​ colored basic fuchsin.
A good quality Schiff's reagent should be made from pararosaniline rather
than basic fuchsin, i.e. the specific component of basic fuchsin which produces
the brightest staining solution. Beware of dye batches which give a brown
solution when first made. Only a water clear or pale amber solution is
acceptable. Although this brown discoloration may be removed with activated
charcoal, the original depth of brown is indicative of the amount of non-
pararosanilin components present in the dye. If large amounts of charcoal are
needed to remove the brown color, or the charcoal has to be allowed to stay in
the solution for longer than about a minute to make it water clear, then it is
quite possible that the staining abilities of the Schiff's reagent may be
compromised.
The most common application for Schiff's reagent is the Periodic Acid
Schiff, or PAS, reaction. This is a technique for the demonstration of
carbohydrates in tissue sections. The purpose of the periodic acid is to oxidize
some of the tissue carbohydrates. This produces aldehyde groups, which can
then condense with Schiff's reagent forming a bright red coloration and
demonstrating the tissue component to which the carbohydrate is attached.
Schiff reagent can be prepared in different ways: (1) by using thionyl
chloride to release sulfur dioxide; (2) by adding sodium or potassium
metabisulfite; or (3) by using sulfur dioxide gas. All of these methods utilize
the same principle of sulfuration in order to rearrange the chromophore group
that is present in basic fuchsin. Any excess sulfur remaining in solution,
together with any nonspecific yellow dye contaminant that is sometimes
present in fuchsin, is absorbed and removed by subsequent treatment with
activated charcoal.
Schiff reagent is stored in the refrigerator so it is important to allow the
reagent to come to room temperature before use. Failure to do so may result in
weak staining. It is also important to store Schiff reagent in a tightly closed
container when not in use so that the solution remains potent and stable.

Barger and de Lamater Method (1948)
1. Dissolve 1 g. basic fuchsin in 400 ml. boiling distilled water.
(Remove the flask of water from the bunsen burner just before adding
the basic fuchsin, to prevent splashing of solution.)
2. Cool to 50°C and filter.
3. Add 1 ml. thionyl chloride to the filtrate.
4. Stopper, shake, and then let stand in the dark for 12 hours.
5. Add 2 gm. activated charcoal to decolorize, shake for l minute, and
filter into a brown stock bottle.
 6. Store in the dark at 0 to 4°C.
7. Allow the aliquot required to reach room temperature before use.

De Tomasi-Coleman Method (1939)
1. Dissolve l gm. basic fuchsin in 200 ml. of boiling distilled water,
shaking for 5 minutes.
2. Cool to 50°C and filter.
3. To the warm filtrate add 20 ml. of 1 M HCl (98.3 ml. HCl, sp gr
1.16, made to 1000 ml. with distilled water).
4. Cool to 25°C.
5. Add l gm. anhydrous sodium or potassium metabisulfite.
6. Let stand in the dark for 16 to 24 hours (solution will be orange or
straw-colored).
7. Add 2 gm. activated charcoal to decolorize, shake for l minute, and
filter into a brown stock bottle.
8. Store in the dark at 0 to 4°C.
9. Allow the aliquot to reach room temperature before use.
Both Schiff reagents are stable and may last up to 6 months. However, it is
preferable to make fresh reagent every month. The reagent should be discarded
when it begins to form a color. Aside from running control sections when
staining, reactivity of the Schiff reagent may be tested by adding a few drops of
the reagent to 10 cc of 37- 40% formaldehyde. Rapid development of a reddish
purple color means that the Schiff reagent is still good and usable. If the
reaction is delayed and the resultant color is deep blue-purple, the solution is
breaking down and should be discarded.
STAINING OF GLYCOGEN
Glycogen is the main storage form of glucose, manufactured and stored
chiefly in the liver, but is also found normally in less quantity in muscles,
parathyroid and cartilage.
Glycogen is very soluble in water and insoluble in alcohol. Theoretically,
therefore, alcoholic solutions are supposed to be the best fixatives and aqueous
fixatives are not suitable. This is not entirely observed in practice because
glycogen is ultimately lost no matter what fixative is used. Glycogen is often
admixed with protein and lipids and hence is also coated with a protein
membrane during protein fixation, thereby preventing further loss of glycogen.
Furthermore, formalin and picric acid tend to bind glycogen with proteins.
 Although alcohol renders glycogen insoluble, it penetrates the tissues
slowly; hence, preserves only the glycogen on the surface of the block. It also
causes polarization or streaming of glycogen to one end of the cell, causing
glycogen to appear in the form of clusters of large coarse granules along the
margins of the cell.
For adequate demonstration of glycogen, thin slices of tissues should be as
fresh as possible, and should be immediately placed in the fixative at 4°C to
prevent rapid breakdown of glycogen into glucose, due to the action of strong
glycogenolytic enzymes particularly in the liver.
For routine processing, the following fixatives are recommended, i.e.,
acetic acid, alcohol formalin, Bouin's, Brasil's, Kelly's and Gendre's solution.
From the fixative, the tissue blocks should go directly into absolute alcohol. If
minimal loss of glycogen is desired, it is advisable to float the sections onto
75% ethanol when cutting, and also to mount them on slides from 75% ethanol.
Because of its water solubility, rinsing of tissues in normal saline before
fixing should be avoided. For staining, blocks should be fixed in absolute
alcohol to prevent dissolving water soluble glycogen.
A positive control section should always be treated in parallel with the
section being examined, to ensure that the staining technique is working.

PERIODIC ACID-SCHIFF REACTION (McManus 1948, Carson 1983)
Fixation: 10% neutral buffered formalin or Bouin’s solution.
Sections: 4-5 µm Paraffin
Solutions:
Periodic Acid, 0.5% Solution
Periodic acid 2.5 gm
Distilled water 500 ml
1N Hydrochloric Acid
Hydrochloric acid, conc. 83.5 ml
Distilled water 916.45 ml
Add acid to the water and mix well.
Potassium Metabisulphite, 0.55%
Potassium metabisulphite 2.75 gm
Distilled water 500 ml
Schiff Reagent
Distilled water 800 ml
Basic fuchsin 4 gm
Sodium metabisulphite 4 gm
1N Hydrochloric acid 80 ml
 Heat water to the boiling point. Remove from flame, add basic
fuchsin, and again heat solution to boiling point. Cool the solution to
50oC and then filter. Add 80 ml of 1N HCl, cool completely, and
then add 4 gm of sodium metabisulphite. Let the solution stand in
the dark overnight; it should turn light amber. Add 2 m of activated
charcoal and shake for 1 minute. Filter the solution, and store in the
refrigerator. The solution should be stable for 2-4 months.
Method:
1. Bring sections to water.
2. Oxidize in 5 minutes in 0.5% aqueous periodic acid.
3. Wash in running water for 5 minutes and rinse in 3 changes of
distilled water.
4. Place in Schiff's reagent for 10-20 minutes (10 minutes for frozen
sections). Schiff reagent should be allowed to warm to room
temperature before use.
5. Wash for 30 minutes in running water or rinse three times in 0.5%
aqueous sodium metabisulfite, freshly prepared, and wash in running
water for 10 minutes.
(The metabisulfite rinse is optional, to remove excess leucofuchsin
which might regain color on oxidation and cause false positive
staining).
6. Wash in running tap water or 10 minutes to develop full color
7. Optional: Counterstain in Harris hematoxylin with acetic acid (2 ml
acetic acid and 48 ml hematoxylin) for ½ minute.
8. Wash in tap water, "blue" in Scott's tap water substitute, and wash
for 5 minutes in running tap water.
9. Dehydrate in increasing concentrations up to absolute alcohol,
clear and mount in Clarite or Permount.
Results:
PAS-positive substances red or magenta red
Nuclei blue
NOTES:
The method described gives maximum reaction but may produce a
little background coloration. Alcoholic PAS reaction uses buffered
alcoholic solution of periodic acid and reducing sulfite rinse.
Mucoproteins are the most common PAS positive substances, e.g.,
mucin and intestinal mucoid secretions, tracheobronchial aspirates, and
 hyaline casts (kidney). Carbohydrates, glycoproteins, glycolipids,
unsaturated lipids and phospholipids are also PAS​ positive, provided they
are retained in the section. PAS-positive staining can also be found in
certain bacteria, fungi, kerasin, connective tissue mucin, basement
membrane, thyroid and cartilage.
The PAS reaction is a useful indicator for glycogen when the technique
incorporates a diastase digestion stage.

Fig. 18-1. PAS Stain - Liver

PAS with Diastase Method for Glycogen Demonstration
Periodic Acid Schiff (PAS) stain is widely used for demonstration of
glycogen. However, because of the fact that many other substances are also
demonstrated, control sections should be processed simultaneously for proper
evaluation. The section serving as control is treated with diastase (human
saliva) or 0.1% malt diastase in distilled water to remove glycogen. For
practical purposes, PAS reaction with and without diastase is considered as a
specific test for glycogen.
Glycogen is readily digested with amylase. Malt diastase is extracted from
malt and contains both alpha- and beta- amylases. It is the commonly employed
enzyme for glycogen digestion because it is easy to use, stable and
comparatively cheap. Saliva containing ptyalin (salivary amylase) is also a
highly effective means of digesting glycogen in tissue sections. However, most
workers prefer the commercial diastase because it is easier to standardize.
Fixation: Helly's Fluid
Solution:
Diastase 1 gm.
Distilled water 100 ml.
Method:
1 Sections to water.
2. Treat the control with saliva or I % malt diastase in distilled
water.
 3. Wash in running tap water for 5 minutes.
4. 0.5% periodic Acid for 5 minutes.
5. Rinse in distilled water.
6. Schiff's reagent for 5 minutes.
7. Rinse in 3 changes of sulfurous acid for 3 minutes each.
8. Wash in running tap water for 5 minutes.
9. Harris hematoxylin for 30 seconds.
10. Blue" in running tap water for 5 minutes.
11. Dehydrate, clear and mount.
Results:
Nuclei blue
Glycogen red
NOTE:
The diastase digestion step should be performed with solutions that have
been preheated to 37° C for one hour prior to use. The strength of the diastase
solution should be optimized to give complete glycogen digestion.
Concentrations (w/v) of 0.1% – 1.0% are commonly used. The diastase should
be dissolved in a phosphate buffer with a pH of around 6.0 for maximum
effectiveness. Diastase dissolved in water may not give adequate performance.
PAS Technique with diastase control is the method of choice for glycogen
staining. Substances that are positive to Schiff's reagent without periodic acid
oxidation are not necessarily carbohydrates. Hence, controls should be included
to show substances that are only positive to Schiff after specific oxidation.
Other methods used for staining glycogen are:
1. Best Carmine Method
2. Langhan's Iodine stain

Best Carmine Method (Modified Sheehan 1980, Luna 1992)
Best Carmine is a good staining technique due to the affinity of alkaline
carminic acid for glycogen, producing a bright red color. Ehrlich's hematoxylin
is used as a counterstain, coloring the nuclei blue. Although carminic acid is
believed to be the essential ingredient, carmine is generally used in its place
because the acid form is more expensive and not necessarily more effective.
Potassium carbonate and potassium chloride salts are added to the stock
solution to inhibit any non-specific background carmine staining due to
electrostatic bonding between the negatively charged carmine and the basic
proteins found in tissue.
This method is selective but not as highly specific for glycogen as the PAS
method with and without diastase. Mast cell granules, fibrin and mucin are also
stained, albeit weakly.
Fixation: 10% neutral formalin, absolute alcohol, formol-saline, or
Carnoy's fluid.
Sections: Celloidin sections are the best. Paraffin sections should be
covered with a thin layer of celloidin before staining (celloidinization)
to prevent the loss of sections from the slide due to the ammoniacal
staining solution, and to preserve glycogen.

Celloidinization of Slides
1. Sections to absolute alcohol.
2. Place sections in 1% celloidin in equal parts of absolute ethyl
alcohol and ether for 5 minutes.
3. Wipe dry the back of the slide and without allowing celloidin to
dry, transfer the slide to 80% ethyl alcohol for 5 minutes.
Solutions:
Carmine Stock Solution:
Carmine 2 gm
Potassium carbonate 1 gm
Potassium chloride 5 gm
Distilled water 60 ml
Boil gently for 5 minutes, using a large flask to avoid spilling.
Cool and add 20 ml. concentrated ammonia. Filter and store in a dark
 container at 4°C. (The solution should be usable for several months.)
Carmine Working Solution:
Stock solution 5 ml
Concentrated ammonia 12.5 ml
Methanol 12.5 ml
The working solution should be filtered before use.
Best's Differentiator
Methanol 40 ml
Ethanol 80 ml
Distilled water 100 ml
Method:
1. Rinse celloidinized slide briefly in water.
2 Stain with Harris hematoxylin for 5 minutes or Mayer hematoxylin for
15 minutes.
3. Wash in running water for 15 minutes.
4. Stain in working solution of Best carmine, in a small closed jar for
30 minutes.
5. Differentiate in Best's differentiator for 30 seconds or until no more
color comes away.
6. Rinse rapidly in 80% alcohol, or place immediately in 95% alcohol.
9. Remove or dissolve celloidin film by transferring the section to
alcohol ether mixture.
10. Dehydrate in absolute alcohol, clear and mount.
Results:
Nuclei blue or grayish blue
Glycogen pink to red
Mucin weak red

Fig. 18-2. Best Carmine stain - Liver

NOTES:
Staining may be improved by reducing the amount of ammonia or methyl
alcohol. Celloidin film, if too thick, may become insoluble after Best carmine
staining, particularly if saliva has been used as control. This is quite impossible
to remove completely. In such cases, the stained section should be examined
thru the portion of the film that tends to be lightly stained.
Failure to stain may be due to using old deteriorated stock solution, or to
washing the section in water instead of alcohol or differentiator immediately
after staining with carmine. The section should not be allowed to dry following
staining with carmine, but should be washed immediately in alcohol or
differentiator. Staining should be carried out in a closed container (e.g., Coplin
jar) as the highly volatile ammonia readily evaporates, causing the carminic
acid to precipitate out of the solution.
STAINING OF MUCIN
Mucins are polysaccharides bound to other substances (the nature of which
serves as the basis for their classification), forming the ground substance of
connective tissues primarily. Mucins usually take up a light pink color with
eosin when stained with H&E, unless Ehrlich's hematoxylin (alkaline) is used,
in which case acid mucopolysaccharides especially from connective tissue will
stain bluish or basophilic.
They may be precipitated by dilute acetic acid and dissolved by dilute
alkali; hence, alkaline fixatives are usually contraindicated. Formalin and
Carnoy's fluid are recommended for fixation although prolonged storage in
formalin tends to reduce the strength of PAS reaction of mucin. Hydration with
0.2.N NaOH for 10-15 minutes before staining may be done to regain PA.S
reaction although such procedure almost invariably causes detachment of
sections from the slide.
There are a variety of mucin stains, all attempting to demonstrate one or
more types of mucopolysaccharide substances in tissues. The types of
mucopolysaccharides are as follows:
• Neutral mucopolysaccharides - These substances contain hexoses as
their second carbohydrate component, are less distributed and less
important than acid mucopolysaccharides, and found in epithelial and
intestinal glands.
• Acid (simple, or non-sulfated) - Are the typical mucins of epithelial
cells containing sialic acid. They stain with PAS, Alcian blue at pH 2.5,
colloidal iron, and metachromatic dyes. They resist hyaluronidase
digestion.
• Acid (simple, mesenchymal) - These contain hyaluronic acid and are
found in tissue stroma. They do not stain with PAS, but do stain with
 Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. They
digest with hyaluronic acid. They can be found in sarcomas.
• Acid (complex, or sulfated, epithelial) - These are found in
adenocarcinomas. PAS is usually positive. Alcian blue is positive at pH
1, and colloidal iron, mucicarmine, and metachromatic stains are also
positive. They resist digestion with hyaluronidase.
• Acid (complex, connective tissue) - Found in tissue stroma, cartilage,
and bone and include substances such as chondroitin sulfate or keratan
sulfate. They are PAS negative but do stain selectively with Alcian blue
at pH 0.5.

Acid Mucopolysaccharides
They are polysaccharides with hexuronic acid as secondary carbohydrate
constituent, bound to sulfuric acid esters and proteins. They comprise mainly of
hyaluronic acid (connective tissue mucin usually formed by fibroblasts),
heparan sulfate (usually found in aorta, cardiac connective tissues and mast
cells) and chondroitin sulfate (found in cartilage). They comprise the
intercellular or ground substance all throughout the body, particularly between
connective tissue elements such as reticulum, elastin and collagen. They are
also especially found in synovial fluid, vitreous humor and Wharton's jelly of
the umbilical cord.
Acid mucopolysaccharides are the only large group of carbohydrate
compounds that are not strongly PAS positive. They may, however, be well
demonstrated by the following staining methods:
1. Metachromatic staining
1. Toluidine blue and Azure A
2. Uranyl nitrate- Azure method
2. Alcian blue technique
3. Colloidal iron technique
4. Aldehyde fuchsin stain
5. Mucicarmine stain
6. Fluorescent acridine orange technique

Metachromatic Staining
Acid mucin is metachromatic (producing a color that is different from that
normally exhibited by the dye), while neutral mucin is not. The most useful
metachromatic dye for acid mucin is Azure A. In the Azure A metachromatic
technique, the intensity of staining appears to be due to the initial potassium
permanganate oxidation step which aids in uptake of dye by the tissue. The
alcohol-fastness and increased brightness of metachromasia is due to the
addition of uranyl nitrate that acts as a mordant-type differentiator.
For metachromatic staining, mercurial fixatives are used. Uranyl nitrate
Azure method gives excellent results with connective tissue mucins and is the
only method that is alcohol-fast, allowing permanent preparations to be made
easily.
Toluidine blue is a basic thiazine metachromatic dye with high affinity for
acidic tissue components and nucleic acids. It stains nucleic acids blue and
polysaccharides purple. It is also used to highlight tissue components such as
cartilage or certain types of mucin.
With mucopolysaccharidosis (characterized by abnormal deposition of
mucopolysaccharides or glycosaminoglycans in connective tissues and
hepatocytes), it is necessary to avoid aqueous solutions during fixation,
processing and section preparation. The tissue should be either fixed in an
alcoholic fixation (i.e. Carnoy's fluid) or received fresh for cryostat sectioning.

Metachromatic Toluidine Blue Method
Section: Frozen section
Solution:
0.25% toluidine blue in pH 4.5 buffer
Method:
I. Prepare 15 µm thick fresh cryostat sections (preferably snap-frozen).
2. Stain with toluidine blue solution for 10 seconds only.
3. Blot dry, dehydrate rapidly through absolute alcohol.
4. Clear and mount in a synthetic mountant.
Results:
Mucopolysaccharide red purple
Tissue background blue

Alcian Blue Technique, pH 2.5 (Luna 1968; Carson 1983)
Alcian blue is a histological dye that forms electrostatic bonds with certain
tissue components containing either carboxyl or sulfate groups (such as
sulfated or acid mucins which are specifically and intensely stained. Alcian
blue is the most popular method for general demonstration of acid
mucopolysaccharides, using 3% acetic acid at pH 2.5.
Fixation:
10% neutral-buffered formalin or Bouin’s solution
Sections:
 4-5 µm paraffin sections
Solution:
Acetic Acid, 3% Solution
Glacial acetic acid 15 ml
Distilled water 485 ml
Alcian Blue, 1% Solution
Alcian Blue-8GX 5 gm
Acetic acid, 3% solution 500 ml
Adjust the pH to 2.5, filter, and add a few crystals of thymol
Nuclear-Fast Red (Kernechtrot) Solution
Nuclear fast-red (Kernechtrot) 0.5 gm
Aluminum sulfate 25 gm
Distilled water 500 ml
Dissolve the aluminum sulfate in the distilled water, and then add
the nuclear-fast red. Heat the solution until the nuclear-fast red has
dissolved. Cool, filter, and add a few grains of thymol as
preservative.
Method:
1. Deparaffinize and hydrate sections to distilled water.
2. Place slides in 3% acetic acid solution for 3 minutes.
3. Place slides in alcian blue solution for 30 minutes at room temperature or
for 15 minutes in a 37oC water bath.
4. Rinse sections briefly in 3% acetic acid solution to remove excess alcian
blue.
5. Wash slides in running tap water for 10 minutes.
6. Rinse in distilled water.
7. Counterstain in nuclear-fast red solution for 5 minutes.
8. Wash in running tap water for at least 1 minute.
9. Dehydrate in 2 changes each of 95% alcohol and absolute alcohol.
10. Clear in xylene and mount in synthetic resin.
Results:
Acid mucins blue
Nuclei red
Avoid celloidinization of slides as the alcian dyes are strongly retained
by celloidin.

 Fig. 18 - 3. Alcian Blue stain in myxoid chondroma

Combined Alcian Blue-PAS-Hematoxylin Technique for acid and neutral
mucins (Luna 1968; Carson 1983)
This combined technique is useful for demonstrating the presence of any
mucin, especially for separating acid mucins and neutral mucins. The rationale
is that by first staining all acid mucins with alcian blue, those acid mucins
which are also PAS-positive will not react in the subsequent PAS reaction.
Thus, only the neutral mucins will be stained by PAS, while acid mucins will be
stained by alcian blue.
Fixative:
10% neutral-buffered formalin or Zenker’s solution

Sections:
4-5 µm paraffin sections
Solutions:
Acetic Acid, 3% Solution
Glacial acetic acid 3 ml
Distilled water 100 ml
Alcian Blue, pH 2.5
Alcian blue 5 gm
Acetic acid, 3% solution 500 ml
Periodic Acid, 0.5% Solution
Periodic acid 2.5 gm
Distilled water 500 ml
Stock Reducing Rinse Solution
Sodium metabisulphite 10 gm
Distilled water 100 ml
Working Reducing Rinse
Stock reducing rinse 2.5 ml
Distilled water 50 ml
 Prepare just before use.
Schiff Reagent
Distilled water 800 ml.
Basic fuchsin 4 gm
Sodium metabisulphite 4 gm
1N Hydrochloric acid 80 ml
Heat water to the boiling point. Remove from flame, add basic
fuchsin, and again heat solution to boiling point. Cool the solution to
50oC and then filter. Add 80 ml of 1N HCl, cool completely, and
then add 4 gm of sodium metabisulphite. Let the solution stand in
the dark overnight; it should turn light amber. Add 2 m of activated
charcoal and shake for 1 minute. Filter the solution, and store in the
refrigerator. The solution should be stable for 2-4 months.
Method:
1. Deparaffinize sections and bring to water.
2. Place sections in 3% acetic acid for 1 minute.
3. Stain sections in alcian blue for 30 minutes.
4. Wash sections in running tap water, then rinse in distilled water.
5. Place section in 05% periodic acid for 10 minutes.
6. Wash slides in running tap water for 5 minute, then rinse in distilled
water.
7. Place slides in Schiff reagent for 10 minutes.
8. Place slides in reducing rinse for 5 minutes.
9. Wash in running tap water for 10 minutes.
10. Stain sections with Harris hematoxylin containing acetic acid (48 ml
hematoxylin in 2 ml glacial acetic acid) for ½ to 1 minute. (Optional)
11. Dehydrate in 2 changes each of 95% and absolute alcohols clear
in xylene, and mount with synthetic resin.
Results:
Acid mucins blue
Neutral mucins magenta
Mixtures of above the color will range from blue-purple
through purple to a violet or mauve color, depending on
dominant component
Nuclei pale blue
It is important to stain only lightly with hematoxylin to distinguish it from
alcian blue staining. Ehrlich's hematoxylin should be avoided as a
counterstain, as it will also stain some mucins and will complicate the final
result.
 Fig. 18-4. Alcian Blue-PAS Stain
(Acid mucin- blue; Neutral mucin-magenta)

Gomori's Aldehyde Fuchsin Stain
Aldehyde fuchsin was first introduced by Gomori in 1950 as an elastic
tissue stain. It employs basic fuchsin plus aldehyde to demonstrate sulfur​-
containing compounds .The blueing of basic fuchsin is due to its combining
with a mixture of acid-alcohol and paraldehyde, forming acetaldehyde which
then condenses with the amino groups of the basic fuchsin. In the presence of
strong mineral acid, basic fuchsin forms intensely purplish dyes with certain
aldehydes. A number of other tissue components are equally stained, including
acid mucopoly-saccharides, sulfated mucosubstances, pancreatic islets of
Langerhans, thyrotrophic hormones, and secretory substances. It is also used to
stain mast cells, particularly when no counterstaining is done.
Aldehyde fuchsin has a greater affinity for sulfated mucins, which are
stained purple, while the carboxylate forms (sialomucins, e.g. submandibular
salivary gland and intestinal goblet cell) only will be stained blue after
subsequent counterstaining with alcian blue.

Mucicarmine Stain
Adding aluminum hydroxide to carmine (Southgate 1927) has improved
the ability of carmine solution (Mayer 1896) to stain for mucin, probably with
the rationale that aluminum salts in the solution will form a chelate compound
with carmine, which then binds to the mucin-containing tissue. Its large
molecular size also allows the dye complex to penetrate and bind to acidic
mucins but not to other acidic substances such as nucleic acids.
Southgate's mucicarmine technique is useful for staining encapsulated
fungi, e.g. Cryptococcus neoformans.
Preparing the Stain:
1. Grind 1 gm. carmine and place in a large (500 ml.) conical flask.
2. Add 100 ml. of 50% alcohol and mix.
 3. Add 1 g. aluminum hydroxide and mix
4. Add 0.5 g. anhydrous aluminum chloride.
5. Mix and boil gently for 2 Yi minutes.
6. Cool, filter and store at 4°C.
The mucicarmine solution will usually keep for 6 months or so at 4°C
before its stain properties deteriorate. Solutions more than 6 months old
should be discarded.
Method:
1. Deparaffinize section and bring to water.
2. Stain the nuclei with Mayer's hematoxylin (not Ehrlich's
hematoxylin). Differentiate well and blue in the usual way.
3. Stain with mucicarmine for 20 minutes.
4. Wash in water. Rinse in absolute alcohol.
5. Clear in xylene and mount.
Results:
Mucins red
Nuclei blue
Background unstained
NOTE: Ehrlich's hematoxylin should be avoided as a counterstain
because certain mucins pick up the hematoxylin and consequently will
not be stained. As a general mucin technique, the combined alcian
blue-PAS technique will be more informative in establishing the
presence or absence of tissue mucins.

Fig. 18-5. Cryptococcus Neoformans (Mucicarmine stain)

Colloidal (Dialyzed) Iron Technique
The colloidal iron technique works on the principle that at a low pH,
colloidal iron will be adsorbed onto tissue containing acid mucins, and
subsequently visualized by conversion to ferric ferrocyanide (prussian blue)
using the conventional Perl's technique. The dialyzed iron-prussian blue can be
combined with PAS reaction to give color differentiation between acid and
neutral mucins. This has greater sensitivity and intensity of reaction compared
to alcian blue staining, but is a more complex and time-consuming method.
Iron particles are stabilized in ammonia and glycerin and are attracted to acid
mucopolysaccharides. It requires formalin fixation. Phospholipids and free
nucleic acids may also stain. The actual blue color comes from a Prussian blue
reaction. The tissue can be pre-digested with hyaluronidase to provide more
specificity.

Fluorescent Acridine Orange Technique
Acridine orange is a fluorescent stain (fluorochrome) that can be used to
demonstrate acid mucins. Mucin in a section stained with iron hematoxylin and
acridine orange gives a selective brilliant orange fluorescence, while other
fluorescence in the tissue are subdued by iron hematoxylin and are therefore
colored black. The disadvantage is that fluorescent staining is temporary and
will only last for about 2 hours once the section is mounted.
Fixation: Formalin and other fixatives, except heavy metals.
Sections: Frozen or paraffin Sections
Method:
1. Sections to water.
2. Place in 4% aqueous iron hemalum for 5-10 minutes.
3. Wash briefly in running tap water.
4. Stain in 0.1% aqueous acridine orange for 1% minutes.
5. Wash briefly and mount in glycerin.
6. Examine immediately in a fluorescence microscope.
Results:
Acid mucopolysaccharides Black
Fungi Greenish red flourescence
Background Reddish orange fluorescence

Neutral Mucopolysaccharides
They can be found in glands of the GI tract (notably esophagus and
stomach) and in prostate. They stain red with PAS and can be distinguished
from acid mucopolysaccharides because they do not stain with Alcian blue,
colloidal iron, mucicarmine, or metachromatic dyes. The neutral
mucopolysaccharides are glycol proteins poor in sulphuric and uronic acids,
containing 20–50% protein. Up to 80 per cent of the polysaccharides in human
cervical mucus from the midcycle consists of a neutral mucopolysaccharide
which contains as constituents methylpentose, galactose, and hexosamine.
The combination of the alcian blue and the PAS techniques can be used as
a means of distinguishing neutral mucins from acid mucins. In most protocols,
sections are stained with the standard alcian blue (pH 2.5) method followed by
the PAS technique. The alcian blue at a pH of 2.5 will stain all acid mucins
deep blue but will not color the neutral mucins. The subsequent application of
the PAS technique will stain the neutral mucins bright magenta. Tissues or cells
that contain both neutral and acidic mucins may demonstrate a dark blue or
purple coloration.

REFERENCES
Bancroft JD. (1975) Histochemical Techniques. London, Butterworths.
Bancroft JD, Cook HC. (1994) Manual of Histochemical Techniques and their Diagnostic Application.
Churchill Livingstone, Edinburgh.
Carson FL, Pikett JP. (1983) Histochemistry. In: Race GJ, ed. Laboratory Medicine, Philadelphia, PA:
Harper & Row.
Carson FL. (1997) Histotechnology. A Self-Instructional Text. 2nd ed. Chicago: American Society of
Clinical Pathologists.
Charman J, Reid L. (1972) The effect of decalcifying fluids on the staining of epithelial mucins by
alcian blue. Stain Technology, 47: 173.
Clark G. (1981) Staining Procedures (4th ed). Baltimore, Williams and Wilkins.
Cook HC. (1959) A comparative evaluation of the histological demonstration of mucin. Journal of
Medical Laboratory Technology, 16: 1.
Culling CFA. (1963) Handbook of histopathological techniques Ed. 2 Butterworths, London, UK.
Culling CFA. (1974) Handbook of histopathological and histochemical techniques Ed. 3. Butterworths
London, UK.
Dapson RW. (2005) Dye-tissue interactions: mechanisms, quantification and bonding parameters for
dyes used in biological staining. Biotech Histochem 80(2):49-72.
Dapson RW. (2007) The history, chemistry and modes of action of carmine and related dyes. Biotech
Histochem 82:173–187.
Drury RAB, Wallington EA. (1967) Carleton’s Histological Technique (4th ed.) New York, Oxford
University Press, 1967.
Goldstein DJ, Horobin RW. (1974) Rate factors in staining with alcian blue. Histochemical Journal, 6:
157.
Gomori G. (1950) A new histochemical test for glycogen and mucin. American Journal of Clinical
Pathology 16: 177.
Grocott RG. (1955) A stain for fungi in tissue sections. American Journal of Clinical Pathology, 25:
975.
Hale CW. (1946) Histochemical demonstration of acid mucopolysaccharides in animal tissue. Nature
(London), 157: 802.
Hicks JD, Matthaei E. (1958) A selective fluorescence stain for mucin. Journal of Pathology and
Bacteriology, 75: 473.
Itikawa 0, Oguru Y. (1954) Simplified manufacture and histochemical use of the Schiff reagent. Stain
Technology, 29: 9.
Kiernan JA. (2008) Histological and Histochemical Methods: Theory and Practice. 4th ed. Bloxham,
UK: Scion. Chapter 11, pp. 274-306).
Kiernan JA. (2010) Carbohydrate histochemistry. Connection (Dako scientific magazine) 14:68-78.
Lane RF, Tripp EJ. (1971) Basic fuchsin and the preparation of Schiff's reagent. Med Lab Technol.
28(1):26-34.
Lillie RD. (1954) Histologic Technic, 2nd ed., McGraw-Hill, New York and Maidenhead.
Lillie R.D. (1969) H.J. Conn s Biological Stains. Williams & Wilkins, Baltimore.
Luna LG. (1968) Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed., New
York, NY; McGraw-Hill Book Co.
Luna LG. (1992) Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts.
Gaithersburg, MD: American Histolabs Inc.
McManus FA. (1948) Histological and histochemical uses of periodic acid. Stain Technol 23:99.
McManus JFA, Mowry RW. (1960) Staining Methods: Histologic and Histochemical. New York:
Hoeber (Chapter 13, pp. 124-151).
Mowry RW. (1978) Alcian blue techniques for the histochemical study of acidic carbohydrates, Journal
of Histochemistry and Cytochemistry, 4: 407.
Pearse AGE. (1968) Histochemistry: Theoretical and Applied (3rd ed.), Vol. 1. London, Churchill
Livingstone.
Pearse AGE. (1972) Histochemistry: Theoretical and Applied, Churchill Livingstone, Edinburgh.
Pearse AGE. Histochemistry (1985) Theoretical and Applied, 4th ed. Vol. 2. Analytical Technique.
Edinburgh: Churchill-Livingstone, pp .675-785.
Reid H, Clamp JR. (1978) The biochemical and histochemical nomenclature of mucus. British Medical
Bulletin 34: 1.
Rosai J. (2004) Special techniques in surgical pathology, in Rosai J (ed): Rosai and Ackerman’s
Surgical Pathology (9th ed). Philadelphia, PA, Mosby pp 38–41.
Scott JE, Dorling J. (1965) Differential staining of acid glycosaminoglycans (mucopolysaccharides) by
alcian blue in salt solutions. Histochemie 5: 22.
Schulthorpe HH. (1978) Metachromasia. Medical Laboratory Sciences, 35: 365.
Sheehan DC, Hrapchak BB. (1980) Theory and Practice of Histotechnology, 2nd ed. Columbus OH:
Battelle Press.
Southgate HW. (1927) Note on preparing mucicarmine. Journal of Pathology and Bacteriology 30:729.
Spicer SS, and Meyer DB. (l960) Histochemical differentiation of acid mucopolysac-charides by means
of combined aldehyde fuchsin-alcian blue staining. American Journal of Clinical Pathology 33: 453.
Sheehan DC, Hrapchak BB. (1980) Theory and Practice of Histotechnology (2nd ed). St. Louis, MO,
C.V. Mosby Company, 1980.


CHAPTER 19
STAINING OF LIPIDS

In histotechnology, the word lipid refers to all fat and fat like, or fat
containing substances. This includes triglycerides, fatty acids, lipoproteins and
glycolipids. Lipids or fats are generally classified into simple lipids,
compound lipids, and derived lipids. They all have a common property of
solubility in organic solvents and insolubility in water.
1. Simple lipids (neutral fat) are esters of fatty acids with alcohols
and are usually found in the body as energy stores in adipose tissue.
Triglycerides are esters of fatty acids with glycerol constituents,
serving as storage fats in animals with high solubility for certain non-
ionic colored substances (lysochromes) stainable by Sudan Black B,
Sudan IV and Oil Red 0.
2. Compound lipids consist of a fatty acid, an alcohol and one or
more other groups such as phosphorus or nitrogen. They are generally
found in the central nervous system.
a. Phospholipids are the important components of cellular
membranes particularly found in mitochondria and nervous tissue
elements and are readily stained by Sudan Black B and acid
hematin.
b. Glycolipids are composed of fatty acids and hexoses,
possessing characteristics of both lipids and carbohydrates and are
therefore stained by Sudan Black B and PAS techniques.
3. Derived lipids are fatty acids that are derived from hydrolysis of
simple and compound lipids. Examples are cholesterol, bile acids, sex
hormones and adrenocortical hormones.

Adipose Tissue
Adipose tissue or fat is distributed throughout the body in distinct “white”
and “brown” adipose tissue depots. White adipose tissue (WAT) is largely
composed of unilocular lipid-filled adipocytes that specialize in lipid storage,
whereas brown adipose tissue (BAT) is largely composed of multilocular
adipocytes that specialize in lipid burning. Fat cells appear as “signet rings” on
H&E stain because large lipid droplet displace the nucleus and remainder of the
cytoplasm to the edge of the cell. With standard methods of fixation, lipids are
largely lost from tissues during processing. The clear areas in the section are
truly empty, because the triglycerides in the fat droplet are soluble in the organic
solvents used during processing of the section, and are therefore removed from
the section.

Fig. 19-1. Adipose tissue or Fat (H&E stain)

Lipid bodies, also named lipid droplets or adiposomes, are distributed in the
cytoplasm as roughly spherical organelles lacking a delimiting classical bilayer
membrane, but surrounded by an outer monolayer of phospholipids, which at
least in some cells may have a unique fatty acid composition. Cytoplasmic lipid
droplets are neutral lipids, usually triacylglycerols or cholesteryl esters. Because
lipid bodies can be destroyed by drying or fixation and staining with alcohol-
based reagents, there are consequently some methodological limitations to their
study.
Lipids are difficult to demonstrate histologically because they dissolve in
the solvents used for paraffin processing and, to a lesser degree, in celloidin
processing. Frozen sections may be required in order to stain for lipids.
Triglycerides are always completely removed in properly cleared and paraffin
infiltrated blocks. It should be noted, however, that lipids bound to other
materials, such as lipoproteins, lipofuscins and myelin may not be completely
removed by solvents used during paraffin processing and significant amounts
may still be present in the tissue sections.
Lipids are best demonstrated on cryostat sections of fresh unfixed tissue
since there is no really good fixative available. Formalin only preserves those
lipids that are already more or less firmly bound to proteins (such as lipofuscins
and granules of leukocytes). Lipochrome (lipofuscin) pigments are the
breakdown products within cells from oxidation of lipids and lipoproteins.
They are the wear-and-tear pigments found most commonly in heart, liver,
CNS, and adrenal cortex. Lipofuscins (lipochrome pigments) are PAS positive
and variably acid fast. They stain with Ziehl-Neelsen. In addition lipofuscin is
Sudan black B and Sudan Red positive. Lipochrome can be demonstrated by
Schmorl's method which also stains for melanin. Lipochrome may also exhibit
a strong orange auto fluorescence in formalin-fixed, unstained paraffin
sections.

Fig. 19-2. Lipochrome (lipofuscin) appearing as brown pigments.

H&E stain of liver

Lipids present in fat embolism, fatty liver and atheroma may be fixed for
staining in paraffin sections by exposing the sections to an emulsion of linoleic
acid and lecithin in 70% ethylene glycol at 56oC for 3 days. These tissues are
then treated with 2% chromic acid at 4°C for 24 h followed by 24 h in 5%
sodium bicarbonate, with appropriate rinsing between solutions. Paraffin
sections of these tissues then stained with a lipid-soluble dye such as Oil Red
O. The demonstration of fat embolism with good quality tissue detail is made
practical by the method, which is convenient and inexpensive.

Phospholipids and neutral fats will be lost during routine dehydration and
embedding unless they are treated with potassium dichromate or osmic acid,
which are the only agents that truly fix lipids. Oxidation of phospholipids by
chromate fixation renders them non-extractable by alcohol, toluene, xylene or
paraffin. However, they both greatly alter the chemical reactivity of the lipids,
which can adversely affect staining. Formol-calcium is the fixative of choice
for lipid histochemistry, and is prepared by adding 2% calcium acetate to 10%
formalin.
To preserve the lipids, polyethylene glycols (carbowaxes) are sometimes
used in lieu of the usual dehydration and embedding process. However, this
technique is not widely utilized, and in most centers, neutral fats are still best
demonstrated in frozen sections of fixed or unfixed tissue.
 Formol-calcium fixed tissue blocks are also suitable for cryostat sections,
and should be mounted on chrome-gelatin coated slides, because fixation
causes the endogenous tissue proteins to lose their adhesive properties.
In general, histochemical techniques are the common methods of choice
for demonstrating lipids in tissue sections. These are usually complemented by
other biochemical techniques and chromatography for specific identification
and possible quantification of the lipid demonstrated by microscopy.

Fat Stains and Sudan Dyes
Sudanophilia is the property of tissues to be stained with fat or oil-soluble
dyes, regardless of the type of dye used, due to their essential lipid nature. The
staining is based on the greater physical solubility of the dye in lipid substances
than in the usual aqueous-alcoholic or acetone-alcoholic medium in which they
are dissolved. Staining with these dyes is regarded as specific for lipids,
especially for simple lipids (neutral fat). Oil soluble dyes are usually divided
into the main groups: 1. Basic Aryl amines with very low water solubility:
a. Sudan Black B - most sensitive lipid stain known
b. Sudan Red VII B
2. B-Naphthols such as the original diazo dyes
a. Sudan III (C.I. No. 26100)
b. Sudan IV (Scharlach B) C.I. No. 26105 - staining fats with a
more brilliant or deeper red color than Sudan III which stains
lipids orange-red.

Sudan dyes are a group of lipid soluble solvent dyes often called
lysochromes. Sudan III was the first of these dyes to be introduced in 1896,
followed by Sudan IV (Scharlach R) in 1901. Sudan III is predominantly used
for staining triglycerides in animal tissues (frozen sections). With the use of
certain solvents, may also be used to stain some protein bound lipids in paraffin
sections. The most sensitive and versatile of all these dyes is Sudan Black B,
which was introduced in 1935. Unlike other Sudan dyes, Sudan Black B stains
phospholipids as well as neutral fats. However, Sudan Black B does not stain
crystalline cholesterol, and free fatty acids tend to be dissolved in the alcoholic
dye bath. These drawbacks can be overcome by pre-treating the tissue with
bromine to make the unsaturated lipids insoluble in organic solvents. The
uptake of Sudan black B by partition from dilute solution is a specific test for
lipid, but in normally fixed tissue most of the structural lipid is ‘bound’ and is
not accessible to the dye.
 For frozen sections, cut tissues about 15 micra thick are usually stained
with. Scharlach R or with Oil Red 0, which stains neutral fats and lipofuscin
well. Oil Red O is used to demonstrate the presence of fat or lipids in fresh,
frozen tissue sections. Oil Red O is a fat-soluble diazo dye, and is classified as
one of the Sudan dyes which have been in use since the late 1800s. Like most
stains used to detect lipids, Oil Red O is not a true special stain, since it can’t
form bonds with lipid components. It is actually a pigment that functions as an
oil-soluble colorant, and the technique represents a physical method of staining.
For general use, 70% alcohol is an adequate solvent for Oil Red 0 and
Sudan black. Containers should always be kept covered except when the tissue
is being placed into and taken out of solution to prevent evaporation,
particularly of their alcoholic component.

Sudan Black Method for Lipids (Suvarna 2013)
Fixation:
Formaldehyde calcium with post-chroming
Section:
Unfixed cryostat sections preferred, or frozen sections post-fixed in formol
calcium

Method:
1. Sections from water to 50% and 70% alcohol.
2. Stain for up to 2 hours in saturated Sudan Black B in 70% ethanol.
3. Place in 70% alcohol for 5 seconds only to remove excess surface
dye. (Longer periods will remove the color).
4. Immerse in 50% alcohol for I minute.
5. Wash in distilled water for 2 minutes.
6. Counterstain with Mayer's carmalum for 2 1/2 minutes.
7. Wash in distilled water for 2 minutes.
8. Mount in aqueous mounting medium.
Results:
Lipids blue black
Nuclei Red

Sudan IV (Scharlach R) Stain for Lipids
Fixation: 10% Formalin
Sections: Frozen sections
Method: Collect 10 µ frozen sections in distilled water.
1. Place in 50% alcohol for 1 minute.
2. Place in 70% alcohol for 1 minute.
3. Immerse in Sudan IV or Scharlach R (Oil Red 0 may be used) for
5-10 minutes.
4. Dip in 70% alcohol for 1minute (Longer periods will remove the
color.).
5. Counterstain with Harris hematoxylin for 2 minutes.
6. Differentiate in 1% acid alcohol until only the nuclei are stained
blue/ black under microscopic control.
7. "Blue" in tap water for 5 minutes.
8. Rinse in distilled water.
9. Mount sections on to slide from distilled water to an aqueous
mounting medium.
Results:
Lipids (mainly triglycerides) - red
Nuclei - blue/black
NOTE:
Scharlach R (Sudan IV) is the most commonly used stain, producing a
rapid and permanent coloration of lipid. The addition of benzoic acid to
the staining solution materially intensifies the resulting color and
prevents deterioration. True fats stain intensely while cholesterol stains
less intensely.

Oil Red 0 Method in Dextrin (modified by Churukian 2000)
Oil Red 0 is closely related to Sudan dyes and was introduced in 1926 by
French. It was popularized in 1943 by Lillie and Ashburn who advocated its use
as a 50 to 60% fresh aqueous dilution of a saturated 99% isopropanol stock
solution. Oil Red 0 stains neutral fats and lipofuscin well. The basis for staining
lipids with an oil-soluble dye lies in its increased solubility in fatty substances as
opposed to the dye solvents which are used in routine tissue processing. The
choice of solvent for this reaction is also critical, since it must be able to extract
excess dye without dissolving the lipid to be stained- propylene glycol is the
preferred solvent for this technique. The end result is that fat and lipids in tissue
sections stain bright red, and nuclei stain blue. Although other stains are
available to help detect the presence of lipids in tissues, the intensity of its red
coloration makes Oil Red O the preferred choice.
Fixation:
Fresh frozen or frozen sections post-fixed in neutral buffered formalin
Sections:
5 µm sections mount on slides, air dry
Solutions:
Dextrin Solution
Dextrin 1 gm
Distilled water 100 ml
Oil Red O stock solution -
Oil Red O 0.5gm
Isopropanol 100ml
Dissolve the dye in isopropanol using gentle heat in water bath.
Oil Red O working solution
Stock Oil Red O solution 30ml
Dextrin 20ml
Allow to stand for a day or more. Stable or months, filter before use.
Method:
1. Place slides directly into filtered 0.5% Oil Red O in dextrin.
Stain for 20 minutes, rinse with running water briefly.
2. Counterstain with Gill II hematoxylin for 20-30 seconds. Rinse
with water, blue, coverslip with aqueous mounting media.
Results
Lipid red
Nuclei blue

Fig. 19-3. Oil-Red-O stain of liver

Osmic Acid Stain for Fat
Osmium tetroxide (osmic acid) is not a dye but is an unstable oxide which
is reduced to a permanent black substance by unsaturated fats and fatty acids. It
is used as a fixative for electron microscopy and in histochemistry, and for
demonstration of unsaturated fats, with the disadvantage that other substances
may also be stained simultaneously. Lipids may be demonstrated by fixing the
tissue in chrome-osmium solutions or by using frozen section. Reduction of
osmium by thiocarbohydrazide highly enhances lipid labeling. After formol/
glutaraldehyde fixation much of the lipid in the tissues is ‘bound’ and does not
take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
Osmium taken up by tissue proteins at neutral pH is only a small fraction
of that taken up by the lipid. Blackening of osmium tetroxide by unsaturated
lipid is too unpredictable to demonstrate lipid in tissues. Direct fixation with
neutral osmium tetroxide is an effective method for visualizing lipid for the
electron microscope, but the poor penetration of tissue by osmium limits its use
for light microscopy. .

Fixation: 10% formalin
Section: Cryostat section
Method:
1. Collect frozen sections at l0 µ in distilled water.
2. Immerse in l % osmium tetroxide in the dark for 12-18 hours.
3. Wash in distilled water.
4. Wash thoroughly in tap water for 3 hours.
5. Counterstain if desired with l % safranin for l minute.
6. Rinse in distilled water.
7. Rinse in 70% alcohol.
8. Mount on to slides.
9. Blot dry.
10. Dehydrate rapidly, clear and mount.
Results:
Nuclei yellow-orange
Fats black

 Fig. 19-4. Osmium Stain, peripheral nerve myelin

Nile Blue Sulfate Method for Fats
Nile blue sulfate is a dye capable of differentiating two lipid classes
simultaneously by the action of its two components: a red oxazone which
dissolves neutral lipids, and a blue oxazine which is basic and reacts with
phospholipids and free fatty acids. Nile blue sulfate can be used as a
preliminary indicator of the type of lipid present in the tissue section.
Nile red is an excellent stain that is present as a minor component of
commercial preparations of the non-fluorescent lipid stain Nile blue. Nile red is
intensely fluorescent and, if proper spectral conditions are chosen, it can serve
as a sensitive vital stain for the detection of cytoplasmic lipid droplets. Nile red
provides resolution of cytoplasmic lipid droplets in tissues equal to, if not
better than, that obtained with the non-fluorescent dye Oil red 0.

Histochemical Methods
Unlike the fat stains described above, histochemical methods involve
chemical reactions with specific groups, radicals or bonds in the lipid molecule.
Many of these methods are utilized mainly for research studies.

Free Fatty Acids
The histochemical demonstration of free acids is based on the observation
that free fatty acids bind heavy metal ions such as copper to form soaps which
can then be stained with Weigert's lithium hematoxylin,
dimethylaminobenzidine rhodamine, or rubeanic acid. Calcium and iron
deposits will also bind with copper, but can be distinguished from lipid by their
persistence in a de-lipidized control section. They can be extracted with either
1% hydrochloric acid (for calcium) or 5% oxalic acid (for iron salts).


Cholesterol
Most of the earlier methods for demonstrating cholesterol were ineffective
unless cholesterol had been oxidized, either chemically with ferric salts or by
long exposure to atmospheric oxygen. An enzymatic method introduced by
Emeis et al in 1977 based on the production of H2O2 from free cholesterol by
cholesterol oxidase. It involves a two-stage procedure in which esterified
cholesterol is initially hydrolyzed to its free sterol by means of a cholesterol
esterase. Cholesterol is then oxidized by a cholesterol oxidase to release
hydrogen peroxide, which reacts simultaneously with diaminobenzidine to
produce an insoluble brown polymer at the site of cholesterol. Cholesterol
esters can be demonstrated as cholesterol after hydrolysis by cholesterol ester
hydrolase and has also been detected with the use of enzyme cholesterol
esterase. These methods are of limited practical use because the reagents are
expensive, and the procedures are too complex for routine light microscopy. An
enzymatic method for the histochemical localization of cholesterol is presented.
It makes possible the localization of free cholesterol, cholesterol esters, or both
and is compatible with routine histological staining procedures.

Cerebrosides
The sulfate esters of cerebrosides (sulfatides) are generally deposited in
brain and other organs of patients with sulfatide storage disease known as
metachromatic leukodystrophy. Cerebrosides and related lipids are stained by the
Periodic Acid Schiff (PAS) method, designed to stain mucopolysaccharides, and
can be distinguished from glycogen by removal with diastase. Staining with
cresyl violet produces a metachromatic orange color on sulfatides, in contrast to
the orthochromatic purple color of other, less acidic myelin lipids.
Toluidine blue is a standard metachromatic dye for acidic polymers, and
imparts a yellow brown or purplish color to sulfatide deposits. The section is
dehydrated with acetone to eliminate the metachromasia induced by less acidic
groups.


Gangliosides
Neurons may have significant ganglioside storage giving rise to cells
suggestive of gangliosidoses, but storage may not be obvious particularly in
subtypes without mental retardation. The stored gangliosides are PAS (+),
sudanophilic (+), and Luxol fast blue (+).

Fig. 19-5. Abnormal metabolism and neuronal storage of fats in

Gaucher’s disease (PAS stain)

 Gangliosides present in storage diseases like Tay-Sach's disease and
Gm1 gangliosidosis are stained with conventional PAS method. They are
distinguished from other glycolipids by their constituents, neuraminic acid and
sialic acid. A modified PAS method reduces the concentration of the oxidizing
agent (periodate) from 1 to 0.01% to stain sialo-groups that are oxidized more
rapidly than other sugar glycoside. This modified PAS stain has been used to
demonstrate gangliosides (the only glycolipids that contain sialic acids) within
neurons in Tay-Sach's disease.

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Tracy RE and Walia P. (2004). Lipid fixation for fat staining in paraffin sections applied to lesions of
atherosclerosis. Virchows Arch. 445(1):22-6. Epub 2004 Jun 2.
Wigglesworth VB. (1957) The use of osmium in the fixation and staining of tissues. Proceedings of the
Royal Society (London), 8147: 185.
Wigglesworth VB. (1988) Histological staining of lipids for light and electron microscope. Biological
Reviews. 63: 3, 417–431.

CHAPTER 20
STAINING OF PROTEINS AND NUCLEIC ACIDS

Protein is the basic component of living cells and is made of carbon,
hydrogen, oxygen, nitrogen and one or more chains of amino acids linked by
peptide bonds. Based on chemical composition, they occur in tissues either as:
1) Simple proteins: On hydrolysis they yield only the amino acids and
occasional small carbohydrate compounds. Examples are: albumins,
globulins, structural proteins, enzymes, histones and protamines. Simple
proteins can be demonstrated in tissue by histologic methods, amino acid
histochemical methods, enzyme histochemical methods, and
immunocytochemical methods.
2) Conjugated proteins: These are simple proteins combined with some
non-protein material in the body to form complex proteins. Examples
are: lipoproteins, mucoproteins, nucleoproteins, glycoproteins, and
phosphoproteins.
3) Derived proteins: These are proteins derived from simple or
conjugated proteins by physical or chemical means. Examples are:
denatured proteins and peptides.

The three types of proteins based on physical configuration are fibrous, globular,
and membrane.
1) Fibrous Proteins form muscle fiber, tendons, connective tissue and
bone. Examples of fibrous proteins are actin, collagen, elastin,
fibronectin, myosin, tau, tropomyosin and tubulin. The organic portion, or
protein fibers, found in connective tissues are either collagen, elastic, or
reticular fibers. Fibrous proteins are often structural, such as collagen, the
major component of connective tissue, or keratin, the protein component
of hair and nails. Collagen fibers provide strength to the tissue,
preventing it from being torn or separated from the surrounding tissues.
Histologic methods involve the demonstration of fibrous proteins based
on the physical configuration of their molecules, rather than their
chemical composition. Fibrous proteins can be demonstrated by selective
staining with small or large molecule dyes (trichrome method), and silver
impregnation (reticulin method), and specific dye-protein interactions
 (e.g., Congo red stain for amyloid).
2) Globular proteins are more water soluble than the other classes of
proteins and they have several functions including transporting,
catalyzing, and regulating. Almost all globular proteins are soluble and
many are enzymes. Globular proteins are found in blood and tissue fluids
in amorphous globular form with very thin or non-existent membranes.
Examples of globular proteins are albumins, alpha globulin, beta
globulin, fibrin, gamma globulin, hemoglobin immunoglobulins and
myoglobin.
3) Membrane Proteins play several roles including relaying signals
within cells, allowing cells to interact, and transporting molecules.
Membrane proteins often serve as receptors or provide channels for polar
or charged molecules to pass through the cell membrane. Examples of
membrane proteins include c-myc, estrogen receptor, glycophorin D,
histones, hydrolases, oxidoreductases, and p53.

NUCLEIC ACIDS
Nucleic acids are usually combined with basic proteins to form
nucleoproteins. They consist of alternate sugar and phosphate groups, with a
nitrogenous base attached to each sugar group. There are two major nucleic
acids. Deoxyribonucleic acid (DNA) contains a 5-carbon sugar deoxyribose, and
is mainly found in the nucleus of the cell. The four nitrogenous bases of DNA
are purines (adenine and guanine) and pyrimidines (thymine and cytosine).
Ribonucleic acid (RNA) is found in the cytoplasm, and to a lesser extent in the
nucleus, particularly in the nucleolus. It contains ribose sugar with attached
nitrogenous bases of purines (adenine and guanine) and pyrimidines (uracil and
cytosine).

Principles of Staining
Staining depends largely on the attachment of dyes to proteins that have
both positively and negatively charged groups. Phosphate groups of DNA also
are important in nuclear staining. A tissue section contains many proteins that
differ in their isoelectric points. At an ideal pH, certain tissue components will
show a relative acidophilia whereas others display a relative basophilia.
If the pH is adjusted outside the range of about 4 - 8, some groups cease to
ionize altogether, and their staining is inhibited almost completely. For carboxyl
groups the relevant pH is about 4 and below. As the pH alters, there is an impact
on staining, but the impact is gradual. When the pH is at 1.5, no carboxyl groups
are involved with staining. However, phosphate radicals are still ionizable at that
pH and nuclear staining can still be done, although it tends to be highly selective.
Adding a very small amount of acetic acid to 1%-2% aqueous solutions of
neutral red, or the addition of borax to methylene blue can sharpen nuclear
staining. This small amount of acid slightly inhibits the ionic staining of
background tissues, making the largely unaffected ionic nuclear staining appear
more prominent. The final pH is usually about 4, depending on the buffering
capacity of other ingredients. This increases the ionization of tissue amino
groups. Similarly, minor adjustments to make solutions more alkaline can be
done with compounds such as sodium tetraborate (borax) or sodium carbonate.
Acidophilic dyes are attracted to acidic substances, such as mitochondria
and collagen which are anionic (negatively charged) at physiologic pH. Many
proteins are acidophilic at physiologic pH. The aniline dye, eosin, is an acid dye
that stains cytoplasm, muscle, and connective tissues various shades of pink and
orange. Eosin is a red or pink stain that is Acidic / Negative. It binds to
acidophilic substances (such as proteins - which are basic and positively
charged).Commonly substituted acid dyes include orange G or phyloxine. Eosin
is an acid aniline dye that will bind to and stain basic structures (or negatively
charged structures), such as cationic amino groups on proteins. Cytoplasm,
muscle, connective tissue, colloid, red blood cells and decalcified bone matrix all
stain pink to pink/orange/red with eosin. For acidic dyes, the dye in question can
often in addition be selective for particular acidophilic components. I.e. a
technique called the Mallory staining technique uses three acidic dyes: aniline
blue, acid fuchsin and orange G, which selectively stain collagen, cytoplasm and
red blood cells respectively.
Most proteins in the cytoplasm are basic because they are positively charged
due to the arginine and lysine amino acid residues. These form salts with acid
dyes containing negative charges, like eosin. Therefore, eosin binds to these
amino acids/proteins and stains them pink. This includes cytoplasmic filaments
in muscle cells, intracellular membranes, and extracellular fibers.
Basophilic dyes are attracted to basic substances, which are cationic
(positively charged) at physiologic pH. Proteins are basophilic at a pH lower
(more acidic) than their isoelectric point. When the environmental pH is below a
protein's isoelectric point, the protein is negatively charged and hence basophilic.
DNA/RNA in the nucleus, and RNA in ribosomes in the rough endoplasmic
reticulum are both acidic because the backbones of nucleic acids are negatively
charged due to presence of phosphate groups. The negatively charged acidic
backbones form salts with basic dyes containing positive charges. Therefore,
dyes like hematoxylin will bind to DNA and RNA and stain them violet.
Proteoglycans are basophilic due to sugars and esterified sulfates which are
negative at physiologic pH. For basic dyes, the reaction of the anionic groups of
cells (these include the phosphate groups of nucleic acids, sulphate groups of
glycosaminoglycans, and carboxyl groups of proteins) depends on the pH at
which they are used.
Hematoxylin (derived from hematein) is not strictly a basic dye, but it is used
with a 'mordant' that makes this stain act as a basic dye that is generally used in
combination with aluminum ions. The mordant (aluminum salt) binds to the
tissue, and then hematoxylin binds to the mordant, forming a tissue-mordant-
hematoxylin linkage. DNA (heterochromatin and the nucleolus) in the nucleus,
and RNA in ribosomes and in the rough endoplasmic reticulum are both acidic,
so nuclear heterochromatin stains blue and the cytoplasm of cells rich in
ribonucleoprotein also stains blue or purple. The cytoplasm of cells with
minimal amounts of ribonucleoprotein tends to be lavender in color. This
difference in staining intensity is useful in differentiating one tissue from
another. Common basic dyes often substituted for hematoxylin include
methylene blue, toluidine blue, thionine, carmine, basic fuchsin, and azure II. It
may be used after any fixation except fixation with osmium tetroxide.
Frequently, basic dyes (methylene blue, toluidine blue, thionine) will react with a
specific tissue component and impart to it a color different from that of the dye
itself. This phenomenon is called metachromasia and the cell or tissue
components that exhibit it are said to be metachromatic.

Hematoxylin & Eosin Stain (H & E)
The staining method involves application of hemalum, a complex formed
from aluminum ions and hematin (an oxidation product of hematoxylin).
Hemalum colors nuclei of cells (and a few other objects, such as keratohyalin
granules and calcified material) blue. The nuclear staining is followed by
counterstaining with an aqueous or alcoholic solution of eosin Y, which colors
eosinophilic structures in various shades of red, pink and orange.
The staining of nuclei by hemalum occurs due to binding of the dye-metal
complex to DNA, but nuclear staining can be obtained after extraction of DNA
from tissue sections. The mechanism is different from that of nuclear staining by
basic (cationic) dyes such as thionine or toluidine blue. Staining by basic dyes
occurs only from solutions that are less acidic than hemalum, and it is prevented
by prior chemical or enzymatic extraction of nucleic acids. There is evidence to
indicate that coordinate bonds, similar to those that hold aluminum and hematein
together, bind the hemalum complex to DNA and to carboxy groups of proteins
in the nuclear chromatin.
The eosinophilic structures are generally composed of intracellular or
extracellular protein. Most of the cytoplasm is eosinophilic. Red blood cells are
stained intensely red.

Histochemical Identification of Proteins
Histochemical methods are used to demonstrate the presence of amino acid
molecules rather than whole protein molecules. They are based upon
identification of specific linkages or groups within the amino acid molecule such
as the protein​ bound amino groups (e.g. in lysine), phenyl groups (e.g. in
tyrosine), disulfides and sulfhydryl linkages (e.g., in cystine and cysteine),
indole groups (e.g., in tryptophan and tryptamine) and guanidyl groups (e.g., in
arginine). Methods for histochemical detection of specific proteins or of
characteristic groups in proteins are few, and some of those presently available
are somewhat cumbersome for routine histological use, or result in formation of
highly unstable color complexes. If the various amino acids in tissues are to be
demonstrated histochemically, it is important to avoid fixatives such as mercuric
chloride which react with amino acid groups. Neutral buffered formol saline is
the most commonly used fixative for amino acid histochemistry.
Many of the microscope slides are prepared using material embedded in
plastic rather than in paraffin. The plastic embedding medium (glycol
methacrylate) is commonly used in histology and pathology because some of the
artifacts (shrinkage and distortion) caused by hot paraffin can be largely avoided.
Furthermore, plastic embedded sections can be cut at 1 or 2 micrometers thick,
allowing for improved visualization of the tissue. Because H&E stains can be
problematic with methacrylate, some of the plastic embedded material is stained
using a combination of substitute dyes that look similar to H&E but without the
problems.
Staining of ribboned epon sections 0.3-2 μ thick with two intense acid dyes,
Aniline Blue Black and Coomassie Brilliant Blue R 250 for light microscopy
allows precise localization of proteins and, because the sections are ribboned,
facilitates three-dimensional visualization of the structures involved. The dyes
may be used in combination with the periodic acid-Schiff reaction and with
autoradiography.

Alkaline Fast-Green Method for Basic Proteins (especially protamines and
histones)
Fast Green is an acid dye that stains basic groups in the tissues, particularly
basic protamines and histones which have higher isoelectrical points than the pH
of the staining solution. All other proteins have lower isoelectric points: their
basic groups are not ionized and therefore will not stain. Trichloracetic acid is
used to remove nucleic acid which would otherwise mask the basic group of
protamines and histones.

Peracetic Acid-Alcian Blue for Cystine and Cysteine
Peracetic Acid oxidizes cystine and cysteine, forming strong cysteic acid
which is stained blue-green by a basic dye. Sakaguchi’s test for arginine uses
NaOH, sodium hypochlorite (Milton's reagent) and pyridine chloroform,
producing orange-red color on objects containing arginine. The mechanism is
not known.
Proteins with enzyme properties can be demonstrated by histochemical
methods, based on their effect on specific substrates. Recently developed
immunocytochemical methods have also been successful in identifying and
localizing specific proteins such as immunoglobulins, enzymes and hormones.



Proteoglycans
Proteoglycans are proteins that are heavily glycosylated. The basic
proteoglycan unit consists of a "core protein" with one or more covalently
attached glycosaminoglycan chain(s). The chains are long, linear carbohydrate
polymers that are negatively charged under physiological conditions due to the
occurrence of sulfate and uronic acid groups. Proteoglycans occur in the
connective tissue and are a major component of the extracellular matrix. The
protein component of proteoglycans is synthesized by ribosomes and
translocated into the lumen of the rough endoplasmic reticulum. An inability to
break down proteoglycans is characteristic of a group of genetic disorders, called
mucopoly-saccharidoses. The inactivity of specific lysosomal enzymes that
normally degrade glycosaminoglycans leads to the accumulation of
proteoglycans within cells causing a variety of disease symptoms, depending
upon the type of proteoglycan that is not degraded.

Alcian Blue-PAS Staining for Proteoglycans
This is a combined method utilizing the properties of both the PAS and
Alcian blue methods to demonstrate the full complement of tissue proteoglycans.
The rationale of the technique is that by first staining all the acidic mucins with
Alcian blue, those remaining acidic mucins which are also PAS positive will be
chemically blocked and will not react further during the technique. Those neutral
mucins which are solely PAS positive will subsequently be demonstrated in a
contrasting manner. Where mixtures occur, the resultant color will depend upon
the dominant moiety.

STAINING OF NUCLEIC ACIDS
Demonstration of nucleic acids depends upon either reaction of the dyes with the
phosphate groups, or production of aldehydes from the sugar (deoxyribose). No
histochemical methods are available to demonstrate the nitrogenous base.
• Feulgen technique - demonstrates sugar
• Methyl green pyronin technique - demonstrates phosphate
• Acridine orange (by fluorescent method)
• Gallocyanin-chrome alum method demonstrates both DNA and RNA
This last staining method does not separate the two nucleic acids since it
stains both DNA and RNA blue, and suitable extraction technique must be used.
DNA is demonstrated by the Feulgen technique which uses 1 M HCl at
60°C to hydrolyze and break the purine-deoxyribose bond, thereby exposing the
aldehydes which are then stained by Schiff's reagent.
RNA is demonstrated by the methyl green-pyronin technique where methyl
green stains the nuclei by binding preferentially and specifically to DNA, while
pyronin binds to RNA and stains the cytoplasm red.
Phosphate groups of DNA and RNA are acidic and combine with
hematoxylin and other basic dyes by salt linkages. Nucleic acids are best
preserved in alcoholic and acidic fixatives (especially Carnoy's fluid that
contains both alcohol and glacial acetic acid). Formalin is an acceptable fixative,
but has only limited reaction with DNA and RNA. Strong inorganic acids such
as nitric or hydrochloric acid will extract nucleic acids, and should be avoided. A
number of routine histological fixatives such as Zenker's, Susa's, and Carnoy's
do not allow specific nuclear staining. The presence of mercuric chloride in
Zenker's and Susa's fluids is apt to introduce a staining artefact since a divalent
metal ion might serve as a link between carboxyl groups and acid dye ions.

Feulgen Staining for Nuclear DNA (Pearse 1968; Carson 1983)
Feulgen stain is a staining technique used to identify chromosomal material or
DNA in cell specimens. Acid hydrolysis removes purine bases from the DNA,
thereby unmasking free aldehyde groups. Feulgen reaction allows DNA in situ to
be specifically stained based on the reaction of Schiff or Schiff-like reagents
with the free aldehyde groups in proportion to the DNA concentration in the cell
which results in the purple staining. RNA is not hydrolyzed by the HCl treatment
and, thus, the reaction is DNA-specific.
The specimen is subjected to warm (60°C) hydrochloric acid, then to Schiff
reagent. Optionally, the sample can be counterstained with Light Green SF
yellowish. Finally, it is dehydrated with ethanol, cleared with xylene, and
mounted in a resinous medium. DNA should be stained red. The background, if
counterstained, is green.
The Feulgen reaction is considered to be specific for DNA. The other nucleic
acid, RNA, does not react the same way either because the acid hydrolysis
causes it to dissolve away, or because of the hydroxyl group present in ribose
which has lost its oxygen in deoxyribose. It is important to note, however, that if
acid hydrolysis is applied for too long, especially at elevated temperature, then
DNA also can be completely removed, and this is a known source of failure in
the technique. It depends on acid hydrolysis of DNA, therefore fixing agents
using strong acids should be avoided.
FIXATION: Zenker’s, Carnoy’s, Formalin, Kelly's, Regaud's, Flemming’s,
and Susa (except Bouin's and Brasil's fixatives due to their acid components
which may hydrolyze nucleic acid in varying degrees and duration). 5µ
paraffin sections of neutral buffered formalin fixed tissue are suitable.
Fixatives containing strong acids should be avoided as this method depends
on the acid hydrolysis of DNA, and acids in some fixatives may pre-
hydrolyze the tissue (such as picric acid in Bouin's aqueous formal-picric-
acetic mixture).
Formula:
Solution A (1 M hydrochloric acid)
Hydrochloric acid (concentrated) 83.5 ml
Distilled water 916.5 ml
Solution B: Schiff's reagent (to prepare)
Boil 200ml of distilled water, remove flask from Bunsen and add 1g
of basic fuchsin.
Allow to cool to 50°C.
Add 2g of potassium metabisulphate while mixing.
Cool to room temperature and add 2ml of concentrated hydrochloric
acid mix and stand in the dark overnight.
Add a large amount of activated charcoal, shake well and filter. The
solution should be clear/pale yellow.
Store at 4°C
Solution C: Bisulfite Solution
10% potassium metabisulfite 10 ml
1M hydrochloric acid 10 ml
Distilled water 180 ml
Method:
1. Deparaffinize sections to water.
 2. Rinse sections in 1 M HCl at room temperature.
3. Place sections in 1 M HCl at 60°C for 8 minutes (for Carnoy or
formalin fixed tissue).
4. Rinse in 1 M HCl at room temperature for 1 minute.
5. Transfer sections to Schiff's reagent for 45 minutes.
6. Rinse sections in bisulfite solution for 2 minutes.
7. Repeat wash in bisulfite solution for 2 minutes.
8 Repeat wash in bisulfite solution (3rd wash) for 2 minutes.
9. Rinse well in distilled water.
10. Counterstain (optional) in 1% light green for 2 minutes.
11. Wash in water.
12. Dehydrate through alcohols to xylene and mount.

Fig. 20-1. Feulgen Reaction

Results:
DNA red purple
Cytoplasm green
NOTES:
1. Specific hydrolysis time should be observed, based on fixative used.
Tissue fixed with formaldehyde vapor should be hydrolyzed for 30 to 60
minutes, while tissue fixed in Zenker solution should be hydrolyzed for
only 5 minutes.

2. After immersing the slide in Schiff's reagent, it is usually flooded with
bisulfite solution before transferring it to water, to remove the excess
Schiff's reagent from the section, while preventing its oxidation back to
basic fuchsin. If a slide is transferred directly to water from Schiff's
reagent, basic fuchsin will form again, producing a false positive result.

Methyl Green-Pyronin method for RNA and DNA (from Bancroft & Cook
1994)
Methyl green-pyronin method uses the basic dyes to produce a differential
staining reaction for DNA and RNA, respectively. Methyl green is highly
selective for DNA, coloring it green due to the binding of anionic phosphate
group with the stain. Pyronin is somewhat specific for RNA, giving it a pinkish
red color. Methyl green-pyronin stain is also utilized to detect the presence of
plasma cells and lymphocytes.
Fixation: 10% Formalin, absolute alcohol, Zenker's fluid, or Carnoy's fluid
Staining solution:
2% methyl green (chloroform washed) 9 ml
2% pyronin Y 4 ml
Acetate buffer pH 4.8 23 ml
Glycerol 14 ml
Mix well before use.
Method:
1. Sections to water.
2. Rinse in acetate buffer pH 4.8.
3. Place in staining solution for 25 minutes. The staining solution used for
plasma cells works better on Zenker fixed materials.
4. Rinse in buffer.
5. Blot dry.
6. Rinse in 93% alcohol, then in absolute alcohols.
7. Clear in xylene and mount with appropriate medium.
Results:
DNA (Chromatin) Green or blue-green
RNA (Nucleoli) Rose-red;
Granules Dark rose-red
Plasma cell cytoplasm Purple

Fluorescent Staining for DNA and RNA
Fluorochromes are fluorescent dyes which emit light or visible radiation
energy when excited by light of shorter wave length, either visible or ultraviolet.
Disadvantages associated with fluorescent labels include the need for a special
light' source, limited morphology because of poor counterstaining, fading of the
signal on storage, and intrinsic tissue auto fluorescence that is increased by
fixation. Several fluorochrome tracers are presently available:
1. Fluorescein is still the most widely used fluorochrome, because of its
wide absorption spectrum and blue light range. Its characteristic apple
green emission is rarely seen as "auto fluorescence" in mammalian
tissue, which is often blue in color.
2. Rhodamine conjugates absorb maximally in green light, exhibiting an
orange-red emission, and are commonly used in two-color techniques.
 3. Acridine Orange is the most commonly used fluorochrome to
demonstrate DNA and RNA in fresh or fixed tissues, combining with
nucleic acids in cells by salt linkages and cohesion. DNA emits a yellow​-
green fluorescence while RNA is stained brick to orange-red. This is
used for screening of cervical smears for cancer cells. While it has been
used in exfoliative cytology and with alcohol fixed tissues, the results are
not permanent and the technique does not work on formalin-fixed
tissues.
4. Acriflavine, as a 0.01% alcoholic solution, can be used as an alternative
to basic fuchsin in Schiff's reagent, for the Feulgen technique of acid
hydrolysis. DNA is stained by a fluorescent yellow color in this Feulgen​-
type reaction.

Immunohistochemistry
Immunohistochemical staining of tissue sections is perhaps the most commonly
applied protein immunostaining technique. While the first cases of
immunohistochemical staining used fluorescent dye, other non-fluorescent
methods using enzymes such as peroxidase and alkaline phosphatase are now
used. These enzymes are capable of catalyzing reactions that give a colored
product that is easily detectable by light microscopy.
Immuno-electron microscopy allows the detection of specific proteins in
ultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g.
gold) can be directly visualized using transmission electron microscopy. While
powerful in detecting the sub-cellular localization of a protein, immuno-electron
microscopy can be technically challenging, expensive, and require rigorous
optimization of tissue fixation and processing methods.

Antigen Retrieval
Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at
room temperature, and nucleic acids (both DNA and RNA) may be recovered
from them decades after fixation. Many antigens can be successfully
demonstrated in formalin-fixed paraffin-embedded tissue sections. However,
some antigens will not survive even moderate amounts of aldehyde fixation.
Under these conditions, tissues should be rapidly fresh frozen in liquid nitrogen
and cut with a cryostat. The disadvantages of frozen sections include poor
morphology, poor resolution at higher magnifications, difficulty in cutting over
paraffin sections, and the need for frozen storage. The detection of many
antigens can be dramatically improved by antigen retrieval methods that act by
breaking some of the protein cross-links formed by fixation to uncover hidden
antigenic sites.

Electron Microscopy
For electron microscopy, the most commonly used fixative is glutaraldehyde,
usually as a 2.5% solution in phosphate buffered saline. These fixatives preserve
tissues or cells mainly by irreversibly cross-linking proteins. The main action of
these aldehyde fixatives is to cross-link amino groups in proteins through the
formation of methylene bridges (-CH2-), in the case of formaldehyde, or by a
C5H10 cross-links in the case of glutaraldehyde. This process, while preserving
the structural integrity of the cells and tissue can damage the biological
functionality of proteins, particularly enzymes, and can also denature them to a
certain extent. This can be detrimental to certain histological techniques. Further
fixatives are often used for electron microscopy such as osmium tetroxide or
uranyl acetate.
Phosphotungstic acid has long been used as a protein precipitant for positive
staining of whole muscle and collagen fibrils. Phosphotungstic acid acts as an
anionic stain for the positively charged groups of protein and has been used
sporadically as a positive stain in tissue sections, usually as an adjunct to other
stains after osmium tetroxide fixation. Phosphotungstic acid in aqueous solution
serves as a single and reliable stain for aldehyde-fixed tissue, with a view to its
potential use in quantitative electron microscopy.
After aldehyde-fixation, treatment with phosphotungstic acid in aqueous
acidic medium produces an intense electron-opaque stain with minimal
distortion of organelles. Mitochondrial matrix, cisternae of the endoplasmic
reticulum, and the Z-band of muscle are densely stained. The intensity of stain
reflects the concentration of protein based on the quantitative reaction of
phosphotungstic acid with the positively charged groups, and can provide a basis
for the use of the method in quantitative electron microscopy, particularly on thin
sections.

Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis (PAGE) is a technique that is widely used to
separate biological macromolecules, usually proteins or nucleic acids, according
to their electrophoretic mobility. Following electrophoresis, the gel may be
stained (for proteins, most commonly with Coomassie Brilliant Blue R-250,
ethidium bromide; or silver stain), allowing visualization of the separated
proteins. After staining, different biomolecules appear as distinct bands within
the gel. It is common to run molecular weight size markers of known molecular
weight in a separate lane in the gel to calibrate the gel and determine the
approximate molecular mass of unknown biomolecules by comparing the
distance traveled relative to the marker.
DNA is demonstrated by the following extraction techniques:
• Digestion Method: Pure deoxyribonuclease will digest DNA and pure
ribonuclease will digest RNA
• Chemical Methods using:
o 10% perchloric acid at 4oC overnight to remove RNA
o Trichloroacetic acid
o Hydrochloric acid
Stains that allow visualization of the protein pattern in the gel.
• Coomassie Stains
The most popular anionic protein dye, Coomassie Brilliant Blue, stains
almost all proteins. Coomassie Brilliant Blue: R-250 (R for reddish)
offers shorter staining times than G-250 (G for greenish). Coomassie
dyes are also the favorite stains for mass spectrometry and protein
identification.
Ethylene Bromide
Ethidium bromide is a sensitive, easy stain for DNA. The major
drawback to ethidium bromide is that it is a potent mutagen. Solutions
must be handled with extreme caution, and decontaminated prior to
disposal. The dye may be run in the gel with the DNA if desired,
eliminating a separate staining/de-staining process, and nondestructive
nature of ethidium bromide staining have made it the standard stain for
double stranded DNA.
• Silver Stains
Silver stains offer the highest sensitivity, although protocols are often
time-consuming, complex, and do not offer sufficient reproducibility for
quantitative analysis. Protein sensitivity to silver stain offers sensitivity
that exceeds that of Coomassie and is equivalent to most fluorescent
stains.

Fluorescent Stains
These stains are ideal for protein study but are more expensive than
Coomassie or silver stains and require either a CCD (charge-coupled
device) camera or fluorescence scanner for gel imaging.

A silver staining technique has been widely used to detect DNA fragments
with high sensitivity on polyacrylamide gels. The conventional procedure of the
silver staining is tedious, which takes about 40–60 min and needs five or six
kinds of chemicals and four kinds of solutions. Eliminating ethanol, acetic acid,
and nitric acid precession before silver impregnation and using minimal AgNO3
dose on polyacrylamide gel results in a golden yellow staining and transparent
background with high sensitivity of the silver-stained DNA.
Silver staining aids the visualization of targets of intracellular and
extracellular cellular components such as DNA and proteins, such as type III
collagen and reticulin fibers in microscopy of histological sections; in
temperature gradient gel electrophoresis; and in polyacrylamide gels. It is also
used in karyotyping. Silver nitrate stains the nucleolar organization region
(NOR)-associated protein, producing a dark region wherein the silver is
deposited and denoting the activity of RNA genes within the NOR. Silver stains
are also employed in applications that previously employed radiolabeling
because they can achieve comparable levels of detection without the
environmental and economic concerns associated with the use of radioisotopes.
Silver stain is more sensitive and stains lower (2-5 ng) concentrations of
single stranded and double stranded DNA 2-5 ng protein, compared to ethidium
bromide (10 ng double stranded DNA) and Coomassie blue (>= 60 ng protein).
Silver staining relies on the reduction of silver cations to insoluble silver metal
by nucleic acids which are deposited in gel, creating a “latent image” that then
becomes visible by soaking the gel in a solution of silver cations and a reducing
agent. The silver granules in the latent image catalyze the further reduction and
deposition of silver from the solution. Bands manifest as dark brown or black
regions which appear before significant background develops. Development of
the bands is stopped by altering the pH of the gel to a point where silver
reduction is no longer favored.

IN-SITU HYBRIDIZATION
The definitive, most sensitive technique for identifying DNA is in-situ
hybridization. The technique uses a labeled complementary DNA, RNA or
modified nucleic acids strand (or probe) to localize a specific DNA, RNA,
nucleic acid sequence or gene expression within a cell, in contrast with
immunohistochemistry, which usually localizes proteins in tissue sections. After
hybridizing a labeled complementary DNA or, a complementary RNA
(riboprobe) to the target sequence at elevated temperature, the excess probe is
washed away (after prior hydrolysis using RNase in the case of unhybridized,
excess RNA probe), and the probe that was labeled with either radio-,
fluorescent- or antigen-labeled bases (e.g., digoxigenin) is localized and
quantified in the tissue either by autoradiography, fluorescence microscopy, or
immunohistochemistry, respectively. The target nucleic acid is retained in-situ
and is not degraded by nucleases, so that it becomes accessible for hybridization
to the probe.
There are two basic ways to visualize the RNA and DNA targets in situ—by
fluorescence (FISH) and by chromogenic (CISH) detection. In situ hybridization
(FISH) multiple targets can be visualized simultaneously and co-localized
multiple gene expression patterns within a single specimen using spectrally
distinct fluorophore labels for each different hybridization probe. Chromogenic
in situ hybridization (CISH) reveals genetic information in the context of tissue
morphology using methods that already available in histology lab. Markers used
to detect the target molecule include isotopes, chromogenic and fluorescent
digoxigenin (DIG). Genes and their transcripts can now be demonstrated by in-
situ nucleic acid hybridization techniques for specific identification of DNA and
RNA sequences. In-situ hybridization has become applicable in routine
laboratories due to the introduction of recombinant DNA technology (by which
sequences of nucleic acids, either DNA or RNA, could be amplified by cloning
and purified to very high levels), and nick translation methods (incorporating
labeled nucleotides into purified DNA and synthesizing a biotinylated
nucleotide) for non-radioactive in-situ labeling.
In-situ hybridization involves the pre-treatment of cellular preparations to
unmask the target nucleic acids, and hybridization of a nucleic acid probe, of
complementary base sequence, to the target cell. This enables specific nucleic
acid sequences or "genes", such as DNA of specific viruses, to be demonstrated
and localized inside individual cells and viewed under the microscope. The
technique is now widely used for the detection of abnormal genes, identification
of viral infection, and for tumor phenotyping.
Nonradioactive in situ hybridization, can be combined with
immunochemistry to identify specific DNA or RNA molecules with fluorescent
probes or tags that can be used for immuno-fluorescence and enzyme-linked
fluorescence signal amplification (especially alkaline phosphatase and tyramide
signal amplification).
DNA in the sample is easy is a highly stable molecule and can be easily
preserved in the sample for subsequent in-situ hybridization. On the other hand,
preserving RNA is much more difficult due to presence of RNase enzyme that
may be found on glassware, in reagents and on the operator and their clothing.
RNase will quickly destroy any RNA in the cell or the RNA probe itself.
Therefore, users must ensure they use sterile techniques, gloves, and solutions to
prevent RNase from contaminating and destroying the probe or tissue RNA.
For good results, older slides should not be stored dry at room temperature.
They should be stored either in 100% ethanol at -20°C, or in a plastic box
covered in saran wrap at -20°C or -80°C. Slides stored in this way can be used
for several years.
RNA probes should be between 250 to 1500 bases in length. Probes
approximately 800 bases long exhibit the highest sensitivity and specificity.
Ideally transcription templates should allow for transcription of both probe
(antisense strand) and negative control (sense strand) RNAs. DNA probes can
also provide high sensitivity for in situ hybridization. However, they do not
hybridize as strongly to the target mRNA molecules as RNA probes. Therefore,
formaldehyde should not be used in the post hybridization washes when using
DNA probes.

Fig. 20-2. Localization by in situ hybridization of amplified ribosomal DNA

Specificity of the probe is extremely important. If the exact nucleotide
sequence of the mRNA or DNA in the cell is known, a precise complementary
probe can be designed. If over 5% of the base pairs are not complementary, the
probe will hybridize only loosely to the target sequence. This means the probe is
more likely to be washed away during wash and detection steps and the probe
may not be detected, or only some of the sites may be detected and the labeling
will not be an accurate representation.

Polymerase Chain Reaction (PCR)
In-situ hybridization techniques are now also supplemented by the
polymerase chain reaction (PCR) method whereby a single copy or a few copies
of a piece of DNA can be amplified across several orders of magnitude,
generating thousands to millions of copies of a particular DNA sequence. In-situ
PCR potentially provides a means of detecting single copies of nucleic acid
sequences in cellular preparations.
The method relies on thermal cycling, through cycles of repeated heating
and cooling of the reaction for DNA melting and enzymatic replication of the
DNA. Primers (short DNA fragments) containing sequences complementary to
the target region along with a DNA polymerase, which the method is named
after, are key components to enable selective and repeated amplification. As
PCR progresses, the DNA generated is itself used as a template for replication,
setting in motion a chain reaction in which the DNA template is exponentially
amplified.
Three major steps are involved in a Polymerase Chain Reaction. These three
steps are repeated for 30 or 40 cycles and done on an automated cycler, a device
which rapidly heats and cools the test tubes containing the reaction mixture.
Each step -- alteration of structure (denaturation), annealing (joining), and
extension takes place at a different temperature: 1) Denaturation - The DNA is
heated at 95oC, breaking the weak hydrogen bonds that hold the double-stranded
DNA, thereby creating two separate single stranded DNA.
2) Primer Annealing - At medium temperatures, around 54oC, the
mixture is cooled, allowing primers to bind (anneal) to their
complementary single-stranded DNA "template" (The template is the
sequence of DNA to be copied.) On the small length of double-stranded
DNA (the joined primer and template), the polymerase attaches and
starts copying the template.
3) Extension - The reaction is then heated to 72oC, the optimal
temperature for DNA polymerase to act, and DNA building blocks
complementary to the template are coupled to the primer, making a
double stranded DNA molecule and adding nucleotides onto the primer
in a sequential manner, using the target DNA as template.
With one cycle, a single segment of double-stranded DNA template is
amplified into two separate pieces of double-stranded DNA. These two pieces
are then available for amplification in the next cycle. As the cycles are repeated,
more and more copies are generated and the number of copies of the template is
increased exponentially. The cycles are done on an automated cycler, a device
which rapidly heats and cools the test tubes containing the reaction mixture.
The PCR amplification of target nucleic acid sequence is more sensitive
than other hybridization methods, but may produce false positive results due to
contamination and non-specific priming.

Reverse transcription polymerase chain reaction (RT-PCR), one of many
variants of polymerase chain reaction (PCR), is the most sensitive technique for
mRNA detection and quantitation currently available.
In RT-PCR, the RNA template is first converted into a complementary DNA
(cDNA) using a reverse transcriptase. The cDNA is then used as a template for
exponential amplification using PCR.
The first step in RT-PCR uses reverse transcriptase and a primer to anneal and
extend a desired mRNA sequence. If the mRNA is present, the reverse
transcriptase and primer will anneal to the mRNA sequence and transcribe a
complimentary strand of DNA. This strand is then replicated with primers and
Taq Polymerase, and the standard PCR protocol is followed. This protocol
copies the single stranded DNA millions of times in a small amount of time to
produce a significant amount of DNA.

In Situ PCR
In Situ Polymerase Chain Reaction (In situ PCR) is a powerful method that
detects minute quantities of rare or single-copy number nucleic acid sequences
in frozen or paraffin-embedded cells or tissue sections for the localization of
those sequences within the cells. In this method, the polymerase chain reaction
actually takes place in the cell on a slide, and the product can be visualized in the
same way as in traditional in situ hybridization. Either DNA or RNA can be
detected through the combined examination of PCR/RT-PCR and Histology. The
ability to identify individual cells, expressing or carrying specific genes of
interest in a latent form in a tissue section under the microscope provides a
visual account of silent genes, and allows the determination of various aspects of
normal versus pathological conditions, or latent versus active viral replication.
The principle of this method involves tissue fixing (to preserve the cell
morphology) and subsequent treatment with proteolytic digestion (to provide
access for the PCR reagents to the target DNA). The target sequences are
amplified by those reagents and then detected by standard immunocytochemical
protocols. In situ PCR combines the sensitivity of PCR or RT-PCR amplification
along with the ability to perform morphological analysis on the same sample,
and thus it is an attractive tool in diagnostic applications. One of the most
prominent applications is the detection of infectious disease agents including
HIV-1, HBV, HPV, HHV-6, CMV, and EBV.
Successful in situ hybridization and PCR can be done with unfixed, frozen
tissue or after fixation in acetone, ethanol, or buffered formalin. Fixatives that
include a heavy metal or picric acid do not allow for PCR because of the rapid
and extensive degradation of the DNA. 100% detection of the target is achieved
only when prolonged formalin fixation is followed by protease digestion. PCR in
situ hybridization is best used to detect DNA in paraffin-embedded tissue
sections. As with standard in situ hybridization or PCR, variables that can affect
in situ PCR results include type of fixative and time of fixation, protease
digestion, and the composition of the amplifying solution and oligoprobe
cocktail.

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