DE19963198A1 - Diagnosing neurodegenerative disease, e.g., Creutzfeld-Jakob disease and Bovine Spongiform Encephalopathy, on formalin-fixed tissue sections comprises spreading and cleaning a tissue sample on membrane for incubation with protease solution - Google Patents

Abstract

A tissue section (I) is spread out on the upper side of a membrane (II). The paraffin wax is removed and (I) is dried on (II), then incubated with a protease solution (III). An absorbent layer soaked with (III) is brought into contact with the underside of (II). Periodically, (III) is trickled over (I), or coated over it. (II) is washed and finally incubated with protein denaturing agent. Independent claims are also included for the following: (1) a membrane with proteins from a tissue section arranged on its upper surface. Topological arrangement of proteins on the membrane corresponds with the original topological arrangement in the tissue section, the membrane having been prepared as described; and (2) an apparatus comprising a casing (4), base (8) and optional removable cover (6). On the base (8) a layer (10) of absorbent material (12) is arranged, dimensioned for central arrangement with spacing from the wall (14). Its upper surface (18) carries the membrane (20). The protease solution (16) at the base, soaks the absorbent. Preferred features: The casing is trough-shaped. The layer is single or multilayer, comprising the same, or different absorbent materials.

Description

The invention relates to a method for transferring protein to prove this from a formalin-fixed and in Pa neatly embedded tissue section on a membrane with a Top and bottom while maintaining the relative topogra phic positions of the proteins to each other or in the tissue cut.

The invention further relates to a top and bottom having a membrane with a protein arranged on the top from a tissue section.  

The invention further relates to a device for performing a method for converting proteins to their Evidence from a formalin-fixed and embedded in paraffin cut tissue.

The method, the device for its implementation and the Membranes are primarily for use in immune procedures histochemical, semiquantitative, topographic detection of Proteins in formalin-fixed and embedded in paraffin Fabrics provided and suitable.

The immunohistochemical detection of proteins linked to a Membrane bound is both part in the prior art the Western blot as well as part of the histoblot method knows. The Western blot method uses an aqueous solution located proteins first in a polyacrylamide gel according to their running properties separated in an electrical field, in a second step by means of a second electrical Field transferred from the gel onto a membrane and on this membrane in a third step of immunohistochemistry detection reaction (with suitable antibodies) fen. The histoblot method uses a 5-10 µm thin tissue cut from native frozen tissue onto a nitrocellulose Semembran applied and the tissue lysed, whereupon the in the Tissue-containing proteins are released from it and attached attach to the nitrocellulose membrane. Using an immunohisto chemical detection reaction becomes the topographical distribution of the proteins to be detected can be recognized in the tissue context.

The histoblot method has compared to the western blot method the advantage that the proteins on the one hand have their natural to graphic position in the tissue can be assigned and on the other hand through detachment from the tissue and immobilization on a nitrocellulose membrane further chemical (pre) Treatments, especially digestion reactions can be a more sensitive detection in the course of the enable munhistochemical detection reaction.  

However, both known methods are not suitable for the topographic and specific evidence of denatured Proteins in formalin-fixed and embedded in paraffin Tissues. The formalin fixation and embedding in paraffin is but for years the standard method of preserving bi Surgical and autopsy specimens in pathology. That means: for them The vast majority of archived tissue samples exist so far except for the conventional immunohistochemistry no protein detection method, the sensitivity, specificity and topography of the the proteins in question gives satisfactory results.

The object of the present invention is therefore the readiness development of a method that requires the implementation of a sensitive and topographic detection of specific proteins in Tissues that have been fixed in formalin and in paraffin are embedded.

One solution to this problem is to provide a method for transferring proteins from a formally infected and paraffin-embedded tissue section onto a membrane with an upper and a lower side while maintaining the relative, topographic positions of the proteins to one another or in the tissue section, that is characterized by the type and sequence of the following process steps:

• a) Placement and spreading of the tissue section on the O top of the membrane,
• b) dewaxing and drying of the tissue section the membrane,
• c) Incubation of the tissue section with a protease solution (peptidase solution, proteinase solution), where
  • a) the protease solution is arranged at a distance from the top of the membrane and the tissue section beyond the underside of the membrane and is brought into contact with the underside of the membrane by means of a layer of absorbent material moistened with the protease solution, and wherein
  • b) the tissue section is only dripped or overlaid with protease solution periodically, ie at intervals,
• d) washing the membrane, and
• e) Incubation of the membrane with protein denaturant.

Then the protein is detected on the membrane with known detection methods.

The membrane is preferably a nitrocel cellulose membrane; but it can also be another suitable membrane be used.

Another solution to the above task is the loading Provision of a membrane with an upper and a lower side te, the proteins arranged on the top from a tissue has trim, and which is characterized in that the topographic arrangement of the proteins on the membrane of the original topographical arrangement of these proteins in Ge weaving cut corresponds, and that the membrane with the above mentioned method according to the invention can be produced or produced represents is.

To carry out the method according to the invention position of the membrane of the invention is a device proposed by the following characteristics le is characterized by the fact that it is a housing with bottom, wall and with or without a removable lid that has on the bottom a layer of absorbent material is arranged, the Au External dimensions are selected so that the layer is approximately medium ger arrangement on the floor is at a distance from the wall, and their surface facing away from the housing base as a carrier is suitable for a membrane, and that on the bottom a protease solution spaced from the surface of the membrane is, which moistens the layer or from the absorbent Ma material of the layer is included.  

As a result, the membrane lying on this layer stands also in contact with the protease solution and will moistened or soaked by this, and the same applies ultimately also for the tissue arranged on the membrane cut.

The layer of absorbent material can be both single-layer and can also be multi-layer, and in the case of several layers, the individual layers from the same or from different suction pads material (ien).

With the inventive method or with herge made membrane it is possible for the first time also in formalin fixed and in paraffin or similar awake embedded tissues not only qua denatured proteins literally but also quantitatively and topographically sen, namely by simply using the membrane according to the invention proteins arranged thereon of any immunohistochemical is subjected to the verification procedure. Because of that he has inventive methods a higher sensitivity than conventional liche immunohistochemical procedures. For this come in particular re all known and known in the prior art munhistochemical detection methods. So that goes in particular the advantage is that now such tissues samples can be examined for certain proteins that had been fixed in formalin many years ago, in paraf fin embedded and archived to date. Such Un Investigations are primarily in the research and diagnosis of neurodegenerative diseases with a deposit of Proteins (proteins) in the brain are accompanied by large Importance. These include in particular prion diseases, such as e.g. B. Creutzfeld-Jakob disease, scrapie (Scrapie), mad cow disease (BSE) and the fatal familial Insomnia (Fetal Familial Insomnia = FFI) or illness like Alzheimer's disease (where there is an accumulation of A beta amyloid is coming in the brain).  

With the method according to the invention, it has only been possible, for the first time, to display the topographical distribution of PrP ^SC (prion protein with the scrapie isoform) in tissue sections of patients suffering from FFI.

Further details of the invention emerge from the following, detailed description based on execution play and the attached drawing.

Here shows:

Fig. 1 an incubation chamber according to the invention for performing the method according to the invention.

The incubation chamber 2 shown in Fig. 1 consists of egg NEM trough-shaped housing 4 with a removable cover 6th At the bottom 8 of the housing 4 , a layer 10 of absorbent material Ma 12 , for example made of cellulose nonwoven, is arranged. This layer 10 can - as shown here - have one layer; but it can equally well consist of several layers, where the individual layers can then consist of the same or different ver absorbent materials. The outer dimensions of the layer 10 are selected such that they can maintain a distance from the housing wall 14 at all of their edges 12 with a central arrangement on the housing base 8 . The bottom 8 of the housing 4 is covered with a (protease solution, peptidase solution, proteinase solution) 16 which also moisturizes the layer 10 or which is absorbed by the absorbent material of this layer 10 . On the surface 18 facing away from the housing base 8 of the layer 10 , a nitrocellulose membrane 20 is arranged, which on its surface 22 , which points away from the layer 10, carries a fabric cut 24 made of formalin-fixed and originally embedded in paraffin and subsequently deprained again. The tissue section 24 abgewand te surface 26 of the nitrocellulose membrane 20 is connected to En zymlösung 16 loaded or wetted layer 10 over the entire surface and is consequently also be of the enzyme solution 16 wets or saturated. The tissue section 24 in turn lies over the entire surface of the nitrocellulose membrane 20 and is likewise in contact with the enzyme solution 16 in this way .

If the tissue section 24 is additionally drizzled or overlaid with enzyme solution 16 , the liquid thus applied migrates through the tissue section 24 to the nitrocellulose membrane 20 , through this to the layer 10 and from there optionally to the free liquid in the edge areas of the Housing 4 between the housing wall 14 and layer 10 . In order to ensure this flow path of the enzyme solution 16 according to the invention, the liquid level 28 of the enzyme solution 16 should always be below the surface 18 of the layer 10 pointing away from the housing base 8 .

example 1 Immunohistochemical (semiquantitative) topographi Detection of proteins in formalin fixed and tissues embedded in paraffin

Of a formalin-fixed tissue embedded in paraffin be a tissue section is made, on a nitrocellulose membrane applied and dried together with this.

The tissue section on the nitrocellulose membrane is removed finished, transferred to the aqueous phase and dried.

The nitrocellulose membrane with the tissue section is covered with a Protease solution (peptidase solution, proteinase solution) incubated. This protease solution is on the side of the nitrocellulose arranged membrane facing away from the tissue section, and it stands with the surface of this side (i.e. with the Ge webeschnitt facing away) either directly or indi rect, namely by means of a layer of absorbent, with the Protease solution moistened material, in contact.

The surface of the nitrocellulose facing the tissue section membrane and the tissue section itself is arranged by the Protease solution or by using this protease solution moist material spaced or held.

The tissue is removed once or several times cut with protease solution drizzled or overlaid, see above  that the flow path of these trickled or overlaid Protease solution through the tissue section to the nitrocellu loose membrane and through this to the damp if necessary th material and through this to the arranged protea solution runs.

To carry out this incubation (the tissue section on the nitrocellulose membrane with a protease solution), an incubation chamber according to FIG. 1 and associated description is particularly suitable.

After sufficient incubation period, depending on the type and the transferring proteins and the type of protease solution depends and is readily ascertainable for a person skilled in the art, the Nitrocellulose membrane washed and then with a protein naturalization agents incubated.

The nitrocellulose membrane is then washed again and is then ready for use, for example and especially for an immunohistochemical detection reaction.

Example 2 Immunohistochemical detection of the scrapie isoform of prion protein (PrP ^5c ) in formalin-fixed and paraffin-embedded tissue

From a formalin fixed, if CJD formic acid is suspected decontaminated and embedded in paraffin (paraffin through impregnated) tissue block is by means of a known to those skilled in the art and common microtome an approximately 5-7 µm thick tissue section cut off and onto a water surface (water temperature approx. 40 ° C). A commercially available nitrocellulose membrane ran (for example from Biorad) with a pore size of, for example, 0.45 µm is placed on a commercially available glass Place the slide and moisten it in a water bath. Instead of the nitrocellulose membrane can also be another suitable membrane ran. The floating tissue section is included the nitrocellulose membrane from the slide Water surface removed.  

Slides and membrane with tissue cut on top warmed on a hot plate at about 50 ° C for about 1 minute so that the tissue section stretches and the residual water between Tissue section and nitrocellulose membrane is preferred vacuumed with cellulose or filter paper. Then will the tissue section on the nitrocellulose membrane and the object wear in the warming cabinet at approx. 55 ° C for at least 30 minutes dried. After drying, the nitrocellulose membrane can with the tissue section lying on it from the slide taken and either treated directly or until Further treatment can be stored.

The nitrocellulose membrane is used for further treatment overlying tissue section is initially dewaxed, for example wise by incubating in xylene for 2 × 5 minutes washing the xylene with 100% vol / vol isopropanol and from then gradually replacing the isopropanol with what by passing through a descending isopropa nol-in-water dilution series. The final stage of dilution series, namely the 100% Aqua bidest, is preferred with a surfactant, for example 0.1% Tween 20.

The tissue section is then ge on the nitrocellulose membrane dries and can then be used either directly in the verification ver subject to driving or stored until further treatment the.

To detect, for example, the scrapie isoform of prion protein (PrP ^5c ), the tissue section on the nitrocellulose membrane is first digested with proteinase K. For this purpose, the nitrocellulose membrane and the tissue lying on it are first soaked in TBST (10 mM TrisHCl, pH 7.8; 100 mM NaCl; 0.05% Tween 20) and then added for about 8 hours in a closed incubation chamber about 55 ° C on a pad made of absorbent material, for example one or more cellulose layers, incubated with a solution of commercially available Proteinase K (e.g. Sigma) in PK buffer (10 mM TrisHCl pH 7.8 ; 100 mM NaCl; 0.1% Brij 35) in the concentration 250 µm / ml. During the incubation period, the tissue section is wetted several times with Proteinase K solution, e.g. B. by dripping or overlaying. The amount of proteinase K solution intended for wetting is so small that the added liquid can be almost completely absorbed by the base, for example the cellulose layer (s), and there is no liquid level that corresponds to the level. in which the nitrocellulose membrane lies with the tissue section. In other words: while the absorbent pad, for example the cellulose layer (s), is or may be wholly or at least partially in the proteinase K solution, the nitrocellulose membrane with the tissue section lying on it always remains above the liquid level of the proteinase K solution is arranged and - except for scheduled wetting - is not covered by the solution. Under these incubation conditions, the proteins mobilized by the enzymatic digestion of the tissue section are transferred (or transferred or "blotted") into the nitrocellulose membrane.

Following the incubation with Proteinase K solution, the Nitrocellulose membrane (including the largely digested tissue cut) 3 × 5 minutes, first washed in TBST and then eats for 10-20 minutes at room temperature in 4 M guanidini um (iso) thiocyanate solution (GdnSCH) in 10 mM TrisHCl pH 7.8 incu to denatu the proteins in the nitrocellulose membrane rieren. The GdnSCH is then washed out with TBST for 3 × 5 minutes and the nitrocellulose membrane in 0.2% casein in TBST as Blocking reagent (for blocking, i.e. occupying non-specific Binding sites for the primary used subsequently ren antibody) for about 30 minutes. Then will the nitrocellulose membrane with a primary antibody that is directed against the prion protein, for example that in Commercially available antibodies BF4 (company dako) for 1-12 hours the incubated. For this the antibody with blocking reagent to the antibody-specific concentration that is in the state of the Technology usually used for Western blot immune reactions is diluted.  

With incubation times of more than 3 hours, this is approx. 4 ° C, otherwise at room temperature.

Following this incubation with the primary antibody the nitrocellulose membrane is washed 3 × 10 minutes in TBST and then with the secondary antibody in that from the manufacturer specified dilution with blocking reagent for 1-12 hours un under the same incubation conditions as the primary anti body incubated. For example, serves as a secondary antibody se in the case of a monoclonal primary antibody from a Mouse coupled with the enzyme "alkaline phosphatase" Goat anti-mouse antibody (e.g. from Dako, D0486), or in Case of a rabbit polyclonal primary antibody a goat anti-rabbit antibody. In any case, the secondary antibodies must be purified to avoid unwanted, unspe avoid background reactions.

To make the immune reaction visible, the nitrocellulose membrane is subjected to a color reaction. For this purpose, the nitro cellulose membrane is first thoroughly washed for 5 × 10 minutes with TBST. The nitrocellulose membrane is then adjusted to a basic pH by incubation for 2 × 5 minutes in NTM (100 mM TrisHCl pH 9.5; 100 mM NaCl; 50 mM MgCl ₂ ). For the actual color reaction, the nitrocellulose membrane is then subjected to a formazan reaction with 45 µl NTB (75 mg / ml) and 33 µl BCIP (50 mg / ml) in 10 ml NTM at 4 ° C for 7-75 minutes (depending on the primary antibody) subject in the dark. Following the staining reaction, the color reagent is washed out with PBS (= phosphate buffer solution) and the nitrocellulose membrane is cleaned of salt residues and formazan residues in distilled water. All reaction steps are carried out on a tumbler.

The detection reaction is evaluated under stereo magnify after drying the nitrocellulose membrane.

For the detection of proteins other than the scrapie isoform of the prion protein (PrP ^5c ), the same method is carried out using other enzymes instead of Proteinase K. In practice, digestion with protease (1 mg / ml; e.g. from Sigma) or double digestion with protease (1 mg / ml) and proteinase K (250 µg / ml) have also proven to be particularly effective Tissue sections containing connective tissue (tissue outside the central nervous system).

Reference list

2nd

Incubation chamber

4th

casing

6

casing

8th

ground

10th

layer

12th

absorbent material

14

Housing wall

16

Digestive enzyme solution

18th

Surface of the layer

20th

Nitrocellulose membrane

22

surface

24th

Tissue section

26

surface

28

Liquid level

Claims (7)

1. Method for transferring proteins for their detection from a formalin-fixed and paraffin-embedded tissue section onto a membrane with a top and a bottom while maintaining the relative topographic positions of the proteins to one another or in the tissue section, characterized by type and sequence of the following process steps:

• a) placement and spreading of the tissue section on the top of the membrane,
• b) dewaxing and drying of the tissue section on the membrane,
• c) incubation of the tissue section with a protease solution, wherein
  • a) the protease solution is arranged at a distance from the top of the membrane and the tissue cut beyond the bottom of the membrane and is brought into contact with the underside of the membrane by means of a layer of absorbent material moistened with the protease solution, and wherein
  • b) the tissue section is periodically drizzled or overlaid with protease solution,
• d) washing the membrane,
• e) Incubation of the membrane with protein denaturant.

2. The method according to claim 1, characterized in that the membrane is a nitrocellulose membrane or another ge suitable membrane is.   3. Top and bottom membrane with on the O proteins arranged on the top from a tissue section, because characterized in that the topographical arrangement of the Proteins on the membrane of the original topographic Arrangement in the tissue section corresponds, and that the membrane is produced with a method according to claim 1. 4. A device for performing a method according to claim 1, characterized in that it has a housing ( 4 ) with a bottom ( 8 ), wall and without or with a removable cover, that on the bottom ( 8 ) a layer ( 10 ) absorbent material ( 12 ) is arranged, the outer dimensions of which are selected so that the layer ( 10 ) is arranged at approximately the center of the bottom ( 8 ) at a distance from the wall ( 14 ), and its surface facing away from the housing bottom ( 18 ) is suitable as a support for a membrane ( 20 ), and that a protease solution ( 16 ) is arranged on the bottom ( 8 ) at a distance from the upper surface ( 18 ) of the membrane ( 20 ), which moistens the layer ( 10 ) or is absorbed by the absorbent material of the layer ( 10 ). 5. The device according to claim 4, characterized in that the housing ( 4 ) is trough-shaped. 6. The device according to claim 4 or 5, characterized in that the layer ( 10 ) is single-ply. 7. The device according to claim 4 or 5, characterized in that the layer ( 10 ) consists of several layers, wherein the individual layers consist of the same or different absorbent material (s).