RAMOS, Jerome S.
04/26/18
MERRERA, Myles Janine T. 9:30-10:30 TTH
MONTANIEL, Princess G.
MUÑOZ, Marian Louie G.
PINEDA, Eunice
Staining Techniques
CONNECTIVE TISSUE
PURPOSE RESULT OF STAINING
1. Gomori’s Silver Impregnation - To stain reticulin, a fibrillary Reticulin fibers: Black
stain extracellular matrix found throughout the
tissues of the body
2. Van Gieson’s stain - Simplest method of differential staining Nuclei: Brownish black
of collagen using a mixture of picric acid Collagen (fibrous connective tissue): Pink, or
and acid fuchsin Deep red
Musce, Cytoplasm, RBC & Fibrin: Yellow
3. Masson’s trichrome stain - Uses dyes in acid solution involving Muscle, RBC, & Keratin: Red
nuclear staining with iron hematoxylin, Nuclei: Blue-black
followed by cytoplasmic staining with a Collagen & Mucus: Blue
red dye
4. Mallory’s aniline blue stain - Used to stain collagen fibers, reticulum, Collagen fibrils, cytoplasm, fibroglia fibrils,
hyaline fibrils, fibroglia fibrils, smooth & axon cylinders & neuroglia fibers: Red
striated muscle fibers and amyloid Elastic fibers: Pale pink or yellow
- Excellent & colorful method of RBC & myelin sheets: Yellow
demonstrating connective tissue fibers
If it is desired to bring out collagen fibrils as
sharply as possible, omit the staining with acid
fuchsin:
Nuclei & cytoplasm: Yellow
Reticulum: Stand out more prominently
5. Azocarmine stain - Heidenhain’s modification of Mallory’s Amyloid connective tissues & mucous colloid:
aniline blue stain, use azocarmine dye for Deep blue
staining Nuclei: Red
- Valuable stain showing minute details of
connective tissue and of renal glomerular
basement membrane
6. Welgert’s elastic tissue stain - Used to stain elastic fibers present in Elastic fibers: Dark-blue or blue-black on a
the skin, ligaments, aorta, arterial elastic clear background
lamina, and lung (Other structures are colored depending on the
counterstain used)
Nuclei: Red with carmine before or after
staining of fibers
7. Verhoeff’s elastic method - Used to stain elastic fibers present in Elastic fibers: Black
the skin, ligaments, aorta, arterial elastic Nuclei: Gray to black
lamina, and lung Collagen: Red
Cytoplasm & muscle: Yellow
8. Orcein (Taenzer-Unna Orcein - Used to stain elastic fibers, especially in Elastic fiber: Dark brown
method) dermatology due to demonstration of the Nuclei: Blue
finest and most delicate fibers found in
the skin
9. Krajian’s technique - Rapid method for staining elastic fibers, Elastic fibers: Bright red
fibrin, and amyloid employing congo red Fibrin & connective tissues: Dark blue
RBC: Orange-yellow
10. MSB technique - Used to stain fibrin, an insoluble fibrillar Nuclei: Blue
protein resulting from polymerization or Erythrocytes: Yellow
enzymatic coagulation of plasma globulin Muscle: Red
and fibrinogen, forming bundles which Collagen: Blue
contract into dense homogenous masses Fibrin: Red
by employing martius yellow C1 10315, (Early fibrin may stain yellow, and very old fibrin
brilliant crystal scarlet C1 16250 and may stain blue)
soluble blue C1 42780
CARBOHYDRATES
PURPOSE RESULT OF STAINING
1. PAS with diastase - PAS Technique with diastase control is Nuclei: blue
the method of choice for glycogen Glycogen: red
staining
2. Best carmine method - This method is selective but not as Nuclei blue or grayish blue
highly specific for glycogen as the PAS Glycogen: pink to red
method with and without diastase. Mast Mucin: weak red
cell granules, fibrin and mucin are also
stained, albeit weakly
3. Langhan’s iodine method - Mast cell granules, fibrin and mucin are Nuclei blue or grayish blue
(Carleston’s Modification) also stained, albeit weakly Glycogen: pink to red
4. Uranyl Acetate Azure A - To stain acid mucopolysaccharides Mucopolysaccharide red purple
metachromatic technique Tissue background blue
5. Fresh frozen azure A - To stain glycosaminoglycan Glycosaminoglycan- red purple
metachromatic staining
6. Metachromatic toluidine - Toluidine blue is a basic thiazine Mucopolysaccharide red purple
blue staining metachromatic dye with high affinity for Tissue background blue
acidic tissue components and nucleic
acids
. It is also used to highlight tissue
components such as cartilage or certain
types of mucin.
7. Alcian blue technique - Alcian blue is a histological dye that Acid mucins blue
forms electrostatic bonds with certain Nuclei red
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.
8. Combined alcian blue PAS - This combined technique is useful for Acid mucins blue
technique demonstrating the presence of any Neutral mucins magenta
mucin, especially for separating acid
mucins and neutral mucins.
9. Gomori aldehyde fuchsin - A number of other tissue components Sulfated mucins- purple
stain are equally stained, including Carboxylate forms - blue
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.
10. Muricarmine stain - Its large molecular size also allows the Mucins red
dye complex to penetrate and bind to Nuclei blue
acidic mucins but not to other acidic Background unstained
substances such as nucleic acids.
MUSCLE AND BONE
PURPOSE RESULT OF STAINING
Muscle fibers – Green
-Demonstrate frozen sections in muscle Ragged areas - Red
1. Modified gomori’s trichrome biopsy Myofibers and connective tissue – different
stain -Detects ragged fibers in mitochondrial shades of Green
myopathy Nuclei – Red purple
Intermyofibrillary network - Red
Myofibrils – Green
-Demonstrate only muscle biopsies and is
Intermyofibrillar material - Bright red
2. Rapid gomori trichrome stain for staining of freshly cut cryostat sections
Nemaline rods, ragged red fibers - Red on a
mounted on coverslips
blue/green background.
Muscle, neuroglia – Blue
- Ideal for demonstrating striated muscle Nuclei - Deep blue
fibers and mitochondria, often without a Cytoplasm - Pale pinkish brown
3. Mallory’s phophotungstic
counterstain Fibrin - Blue
acid hematoxylin -It is used to identify contraction bands, as Collagen - Deep brownish red
seen in contraction band necrosis Coarse elastic fibers - Bluish
Myelin - Blue to blue-gray
-Method recommended for monochrome
photography
4.Heidenhan’s iron hematoxylin Muscle striations, mitochondria, myelin,
- General stain for epon-embedded tissues
method and chromatin are stained grey - black
as well as paraffin and celloidin-
embedded tissues
- Routine staining method for bone Nuclei – Black
5. Lissamine test red tartrazine
histology (muscle striations are well- Muscles, RBC – Red
method shown) Collagen - Yellow
Lacunae and canaliculi - Dark brown-black
6. Schmorl’s picrothionin - Demonstration of thionin precipitate Bone matrix - Yellow or brownish-yellow
method within the lacunae and bone canaliculi Cells - Red
Lacunae and canaliculi filled with air
lamellae - Brown or brownish black lines
- Used for demonstration of lacunae and
7. Ground section preparation
canaliculi in bone
FAT AND LIPID STAINS
PURPOSE RESULT OF STAINING
- For staining unfixed cryostat sections
preferred, or frozen sections post-fixed in
Lipids blue - Black
1. Sudan black method formol calcium
Nuclei – Red
- For demonstration of neutral
triglycerides and lipids
Lipids (mainly triglycerides) - Red
2. Sudan IV - Demonstrates lipids in frozen sections
Nuclei - Blue/Black
- Stains lipids in fresh frozen or frozen Lipid – Red
3. Oil red O method in dextrin sections post-fixed in neutral buffered Nuclei - Blue
formalin
- For demonstration of unsaturated fats Nuclei - Yellow-orange
4. Osmic acid stain
and staining of cryostat sections Fats - Black
- Acts as a preliminary indicator of the type Neutral lipids - Red
of lipid present in the tissue section Phospholipids and free fatty acids - Blue
5. Nile blue sulfate method
- It serves as a sensitive vital stain for the
detection of cytoplasmic lipid droplets
- Demonstrates sulfatide deposits in brain Sulfatide deposits – Metachromatic red –
6. Toluidine blue acetone
and other organs of patients with sulfatide brown or yellow
method for sulfatide storage disease
TISSUE PIGMENTS AND DEPOSITS
PURPOSE RESULT OF STAINING
1. Perl’s Prussian Blue Method - Perl’s Prussian Blue Method is used to Hemosiderin and Ferric salts stain deep
For Hemosiderin demonstrate hemosiderin and is the blue
major stain use to detect iron in the liver. Other pigments retain their natural color
This histochemical method is based on the Tissue and Nuclei stain red (accdg to
unmasking of ferric iron by dilute counterstain)
hydrochloric acid which is a component of
the acid ferrocyanide solution.
- The Ferric ion then reacts with dilute
potassium ferrocyanide solution to
produce an insoluble blue compound,
ferric ferrocyanide (Prussian Blue)
2. Gomori’s Prussian Blue Stain - Gomori’s Prussian blue Stain is used to Iron pigments stain bright blue
For Iron detect loosely bound ferric iron in tissue Nuclei stain Red
sections. This histochemical reaction is Cytoplasm stain pink to rose
sensitive enough to demonstrate even
minute amounts of iron deposits in bood
cells bone marrow and spleen.
3. Turnbull’s Blue Reaction For - Turnbull’s blue is rarely used in routine Hemosiderin stain blue
Ferrous Iron histology since it is used to detect ferrous Nuclei stain red
iron which is less commonly found than
ferric iron. The reaction depends upon the
union of ferrous iron with potassium
ferricyanide to form a blue precipitate of
complex ferrous ferricyanide. Ferrous
salts are stained blue while others remain
unstained.
4. Benzidine Nitroprusside - Benzidine Nitroprusside demonstrates Hemoglobin and some “oxidase” granules
Stain For Hemoglobin And hemoglobin as well as the oxidase in Leukocyte stain dark blue
Oxidase Granules granules. The stain demonstrates the Nuclei stain red
enzyme hemoglobin peroxidase Most other tissue components are faint
(pseudoperoxidase activity) which is pink
reasonably stable and withstands short
fixation and paraffin processing.
5. Staining For Bile Pigments - Bile pigments contain both conjugated Bile pigments stain emerald to blue
and Hematoidin and Modified and unconjugated bilirubin, biliverdin and Muscles stain yellow
Fouchet’s Technique hematoidin, all of which are chemically Collagen stain red
distinct and show different physical **Hematoidin is not likely to show any
properties. The most common method for color change with this method
demonstration of bile pigments is the
Modified Fouchet’s Technique in which
the pigment is converted to green color of
biliverdin and blue cholecyanin by te
oxidative reaction of ferric chloride in the
presence of trichloroacetic acid.
6. Gmelin Technique For Bile - This method shows an identical result Bile pigments appear as yellow brown
And Hematoidin with liver, bile, gallbladder bile and globules
hematoidin. Gmelin’s test if positive, is a Color spectrum from red to purple to green
diagnostic for bile pigments, although
results produced are only temporary.
7. Schmorls’s Ferric - A test to stain for reducing substances in Bile, Lipofuschins and Melanin stain dark
Ferricyanide Method For tissues including melanin, argentaffin blue
Reducing Substances granules, thyroid colloid, keratin, Argentaffin cells and chromaffin stain dark
keratohyalin, and lipofuscin pigments. blue
Ferricyanide is converted to insoluble Thyroid colloid stain dark blue
Prussian Blue in the presence of ferric ions Nuclei stain red
8. Gomori’s Aldehyde Fuchsin - Gomori’s Aldehyde Fuchsin Technique is Lipofuchsin stain purple
Technique For Lipofuchsin for demonstration of Liofuchcin which Background is yellow
may be seen in neurosecretory cells.
Aldehyde fuchsin is thought to be formed
by condensation of aldehyde groups with
free amino groups of basic fuchsin.
9. Mallory’s Fuschin Stain - Mallory’s Fuschin Stain is used for Nuclei stain blue
demonstration of hemofuscin. Hemofuchsin stain red
Hemofuscin has affinity for fuchsin dyes Hemosiderin is unstained
relatively resistant to decolorization with
alcohol after staining. This stain may be
combined with Prussian blue reaction to
give a complete pigment picture in
hemochromatosis.
10. Masson Fontana Technique - Masson Fontana Technique is used for Melanin stain black
routine demonstration of Melanin. This is Argentaffin cell granules stain black
based on Melanin’s intrinsic property of Nuclei stain red
reducing ammoniacal silver solutions
without the use of extraneous reducer.
This property is known as “argentaffin
reaction”
HISTOCHEMISTRY AND OTHER MISCELLANEOUS
PURPOSE RESULT OF STAINING
1. Enhanced Polymer One Step - To conjugate the primary antibody mixed colours, which were distinct and
Staining directly to the label readily discriminated from red and blue
2. Peroxidase – anti-peroxidase - Recommended for use on blood and Stable, dark brown reaction end product
(PAP) Technique bone marrow smears when antigen is present in the tissue
3. Avian Biotin Complex (ABC) - To detect antigen or antibody in tissues greater concentration of enzyme at the
Technique antigenic site and therefore an increase in
signal intensity and sensitivity upon
addition of substrate
4. Direct Immunofluoresecenxe -Direct reaction with a fluorescein- Apple-green fluorescence when fluorescein
Technique for Solid Tissue conjugated antibody specific for the is used as a fluorochrome.
Biopsies material being sought within the tissue Orange-red fluorescence with rhodamine
conjugates.
5. Indirect - Detection of autoantibodies in the Fluorescence of the target antigen
Immunofluorescence patient’s serum, including the anti-
Technique nuclear antibody, antimitochondrial
antibody, and liver-kidney microsomal
kidney
6. Frozen Section - To identify antigens in fresh frozen Bright yellow fluorescence of target cells
Immunocytochemistry sections
7. Frozen Section - To detect antibodies, particularly for the Bright yellow fluorescence of target cells
Immunofluorescence diagnosis of glomerular diseases in frozen
sections of renal biopsies
8. DIrect Immunoperoxidase - used for the demonstration of various The result can be examined under light
Method substances in tissue sections and utilises microscope
labelled or unlabelled antibodies and the
very stable enzyme, horseradish
peroxidase.
9. Paraffin Wax Section - Used as a diagnostic procedure for The result can be examined under light
Immunoperoxidase Technique formalin-fixed, paraffin-embedded microscope
specimen, and immunolabeling that can
be correlated with morphology
REFERENCES:
Bancroft J.D (2008). Theory and Practice of Histological Techniques. Elsevier Health Sciences
Bruce-Gregorios J.H. (2017). HISTOPATHOLOGIC TECHNIQUES. Published by: Jocelyn H. Brucce-Gregorios,MD, U.S.
Edition
