DE19963198B4 - Method for topographical protein detection on formalin-fixed tissue sections - Google Patents

Abstract

Method for the detection of denatured proteins in formalin-fixed and paraffin-embedded tissue sections, characterized in that the proteins from the tissue sections on 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 according to type and order the following process steps are transferred and proven:
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
ci) 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 which is moistened with the protease solution, and wherein
cii) the tissue section is periodically drizzled or overlaid with protease solution,
d) washing the membrane, and
e) Incubation of the membrane ...

Description

The The invention relates to a method for the detection of denatured Proteins in formalin-fixed and paraffin-embedded tissue sections.

The The invention further relates to a top and bottom Membrane with proteins arranged on top, obtained by a The method of claim 1 or 2.

The The invention further relates to a device for carrying out a The method of claim 1 or 2.

The Process, the device for its implementation and the membrane especially for that Use in procedures for immunohistochemical quantitative topographic Detection of - denatured - proteins in tissue sections fixed in formalin and embedded in paraffin provided and suitable.

To the In principle, there were two procedures with which to register Proteins based on a tissue section topographically below Using immunohistochemical detection methods can be detected can, namely classic immunohistochemical detection and the histoblot technique.

In classic immunohistochemical detection, a thin section of tissue fixed in formalin (or comparable fixants) and embedded in paraffin is applied to a glass surface as a support, dewaxed and the target proteins are displayed with an antibody directed against the target protein with a subsequent detection reaction (see, for example, prion protein expression in different species: Analysis with a panel of new mAbs. ZANUSSO, G. et al., Proc. natl. Acad. Sci. USA (1998) 95, 8812-8816 and Normal host prion protein (PrP ^C ) is required for scarpie spread within the central nervous system. BRANDNER, S. et al., Proc. Natl. Aca. Sci. USA (1996) 93, 13148-13151). For the safety of the employees working with the tissue, tissue is decontaminated before embedding in formic acid if a transmissible spongiform encephalopathy (TSE) is suspected.

at the histoblot technique (see for example regional mapping of prion proteins in brain, TARABOULOS, A. u. a. Proc. Natl. Acad. Sci. USA (1992) 89, 7620-7624) becomes a thin one Tissue section from native frozen tissue on a glass slide as transfer medium is applied and from this cut an impression is immediately made on a nitrocellulose membrane made that in a solution soaked is, with the - native - proteins detached from the fabric association can be. The goal is for proteins to get stuck on the nitrocellulose membrane and can be detected there by immunohistochemistry.

The post-published document "The paraffin-embedded tissue blot detects PrP ^Sc early in the incubation time in prion disases" is only provided by experts. SCHULZ-SCHAEFFER, WJ and others, Am. J. Pathol. (2000) 156 (1) 51-56 The practical problem in pathology is that tissue is only rarely preserved, the usual method is to harden tissue in formalin (or comparable fixants) to stop the decomposition process and at the same time to protect employees from infection to have.

The However, known methods are not suitable for topographical and specific detection of denatured proteins in formalin fixed and paraffin embedded Tissues. Formalin fixation and embedding in paraffin has been around since Years the standard method for the preservation of biopsy and autopsy samples in pathology. That For the most archived tissue samples are so far except conventional immunohistochemistry is not a protein detection method that regarding Sensitivity, specificity and topography of the denatured proteins in question satisfactory results supplies.

task the present invention is therefore to provide a Procedure that implementation a sensitive and topographical proof of specific denatured proteins in tissues which have been fixed in formalin and embedded in paraffin.

• a) Platzierung und Ausbreitung des Gewebeschnitts auf der Oberseite der Membran,
• b) Entparaffinierung und Trocknung des Gewebeschnitts auf der Membran,
• c) Inkubation des Gewebeschnitts mit einer Proteaselösung, wobei ci) die Proteaselösung mit Abstand von der Oberseite der Membran und dem Gewebe schnitt jenseits der Unterseite der Membran angeordnet ist und vermittels einer Schicht aus saugfähigem, mit der Proteaselösung durchfeuchtetem Material mit der Unterseite der Membran in Kontakt gebracht wird, und wobei cii) der Gewebeschnitt periodisch mit Proteaselösung beträufelt oder überschichtet wird,
• d) Waschen der Membran,
• e) Inkubation der Membran mit Proteindenaturierungsmittel.

A solution to this problem in connection with the preamble of claim 1 is that the proteins from the tissue sections on a membrane with a top and a bottom while maintaining the relative topographic positions of the proteins to each other or in the tissue section according to type and sequence with the following Process steps are transferred and proven:

• 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, ci) the protease solution being arranged at a distance from the top of the membrane and the tissue section 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 which is moistened with the protease solution, and wherein cii) the tissue section is periodically drizzled or covered with a layer of protease solution,
• d) washing the membrane,
• e) Incubation of the membrane with protein denaturant.

After that the protein is detected on the membrane using known detection methods.

The inventive method proves to be the most sensitive detection method of aggregated or denatured proteins (based on the amount needed for detection used tissue). The method according to the invention is the histoblot, the Western blot (based on the starting amount) and immunohistochemical Detection method clearly superior to a tissue section drawn on glass slides (see method comparison for BSE tests: Arch Virol Suppl. 2000; 16: 173-80).

at the membrane is preferably a nitrocellulose membrane, however, another suitable membrane can also be used.

A further solution the task mentioned is to provide a membrane with proteins from a tissue section arranged on the top, obtained by a method according to claim 1 or 2, thereby is characterized by the topographical arrangement of the proteins on the membrane of the original corresponds to the topographical arrangement in the tissue section.

to execution of the method according to the invention and a device for producing the membrane according to the invention proposed, which is characterized by the features mentioned below distinguishes them as a housing with bottom, wall and without or with removable cover, that a layer of absorbable material is arranged on the floor, whose external dimensions are selected that the layer at approximately centered on the floor at a distance from the wall, and their from the bottom of the case groundbreaking surface as a carrier for one Membrane is suitable, and that at the bottom with a protease solution Distance from the surface the membrane is arranged, which moistens the layer or of the absorbent Material of the layer is included.

Consequently the membrane on top of this layer is also in contact with the protease solution and is or is soaked or soaked by it, and the same ultimately applies to the one arranged on the membrane Tissue section.

The Layer of absorbent Material can be single or multiple layers, and in the case of several layers can individual layers made of the same or different absorbent material (s) consist.

With the inventive method or with the membrane according to the invention produced with it, it is the first time possible, also in formalin-fixed and embedded in paraffin or similar waxes Tissues denatured proteins not only qualitatively but also to prove quantitatively and topographically, namely simply the membrane of the invention with the proteins arranged thereon any immunohistochemical Detection procedure is subjected. This has the inventive method a higher sensitivity than conventional ones immunohistochemical procedures. For this all come in particular State of the art known and common immunohistochemical Verification procedure into consideration. This is particularly advantageous along with the fact that such tissue samples are now also Proteins could be examined which was fixed in formalin many years ago, embedded in paraffin and have been archived to date. Such investigations are in progress especially in the research and diagnosis of neurodegenerative diseases, the one with deposits of proteins (proteins) in the brain accompanied by great Importance. Which includes especially prion diseases, e.g. Creutzfeld-Jakob disease, scrapie, mad cow disease (BSE), and deadly family Insomnia (Fetal Familial Insomnia = FFI) or illnesses such as Alzheimer's disease (where there is an accumulation of A-beta amyloid comes in the brain).

With the method according to the invention, among other things, it was possible for the first time to display the topographic distribution of PrP ^SC (prion protein with the scrapie isoform) in tissue sections of FFI patients.

Further Details of the invention emerge from the following detailed Description using exemplary embodiments and the attached Drawing. Here shows:

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

In the 1 incubation chamber shown 2 consists of a trough-shaped housing 4 With a removable lid 6 , On the ground 8th of the housing 4 is a layer 10 made of absorbent material 12 , for example made of cellulose nonwoven. This layer 10 can - as shown here - be single-layer, but it can also consist of several layers, whereby the individual layers can then consist of the same or different absorbent materials. The outer dimensions of the layer 10 are selected so that they are arranged centrally on the housing base 8th on all its edges 12 a distance from the housing wall 14 can adhere to. The floor 8th of the housing 4 is with a (protease solution, peptidase solution, proteinase solution) 16 which also covers the layer 10 soaked or from the absorbent material of this layer 10 is included. On the bottom of the case 8th groundbreaking surface 18 the layer 10 is a nitrocellulose membrane 20 arranged on their by the layer 10 groundbreaking surface 22 a tissue section 24 made of formalin-fixed material that was originally embedded in paraffin and subsequently deparaffinized. The tissue cut 24 facing surface 26 the nitrocellulose membrane 20 lies with the enzyme solution 16 moistened or moistened layer 10 over the entire surface and is therefore also from the enzyme solution 16 moistened or soaked. The tissue section 24 in turn lies the nitrocellulose membrane 20 over the entire surface and is also in contact with the enzyme solution in this way 16 , Now the tissue section 24 additionally with enzyme solution 16 drizzled or overlaid, the liquid thus applied migrates through the tissue section 24 through to the nitrocellulose membrane 20 , through this to the layer 10 and from there, if necessary, to the free liquid in the edge areas of the housing 4 between housing wall 14 and shift 10 , Around this flow path of the enzyme solution according to the invention 16 to ensure the liquid level 28 the enzyme solution 16 always below that of the case bottom 8th groundbreaking surface 18 the layer 10 lie.

Example 1: Immunohistochemical (Semi-quantitative) topographic detection of proteins in formalin-fixed and tissues embedded in paraffin

Of a formalin-fixed tissue embedded in paraffin made a tissue section, applied to a nitrocellulose membrane and dried along with this.

The Tissue section on the nitrocellulose membrane is dewaxed, into the watery Phase transferred and dried.

The The nitrocellulose membrane with the tissue section is incubated with a protease solution (peptidase solution, proteinase solution). Here is this protease solution placed on the side of the nitrocellulose membrane that corresponds to the tissue section and faces the surface of this side (i.e. with the surface facing away from the tissue section) either directly or indirectly, namely by means of a layer of absorbent, with the protease solution wet material, in contact.

The surface of the nitrocellulose membrane facing the tissue section and the tissue section itself is covered by the protease solution or of that with this protease solution dampened material spaced or held.

On or the tissue section is made several times at intervals with protease solution drizzled or layered, so the flow path this on trickled or layered protease solution through the tissue section to the nitrocellulose membrane and through through this, if necessary, to and through the dampened material runs through to the protease solution.

An incubation chamber according to is particularly suitable for carrying out this incubation (the tissue section on the nitrocellulose membrane with a protease solution) 1 and associated description.

To sufficient incubation period, depending on the species and the species to be transferred Proteins and the type of protease solution depends and for a person skilled in the art can be determined, the nitrocellulose membrane is washed and then incubated with a protein denaturant.

Then will the nitrocellulose membrane is 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 the prion protein (PrP ^5c ) in tissue fixed in formalin and embedded in paraffin

An approximately 5-7 μm thick tissue section is cut from a formalin-fixed tissue block decontaminated with formic acid decontaminated and embedded in paraffin (soaked in paraffin) using a microtome known and known to the person skilled in the art and applied to a water surface (water temperature approx. 40 ° C.) , A commercially available nitrocellulose membrane (for example from Biorad) with a pore size of, for example, 0.45 μm is placed on a commercially available glass slide and moistened in a water bath. Instead of the nitrocellulose membrane, another suitable membrane can also be used. The floating tissue section is made with the Nitrocel lying on the slide Loose membrane removed from the water surface.

Slides and Membrane with a fabric cut on top are placed on a hot plate at approx. 50 ° C for about Warmed up for 1 minute so that the tissue section stretches, and the residual water between the tissue section and nitrocellulose membrane is preferably made with cellulose or filter paper aspirated. Subsequently the tissue section on the nitrocellulose membrane and the slide in the warming cabinet at approx. 55 ° C for at least Dried for 30 minutes. After drying, the nitrocellulose membrane with the tissue section lying thereon removed from the slide and either treated directly or until further treatment be stored.

For further treatment the tissue section lying on the nitrocellulose membrane is first dewaxed, for example, by incubating in xylene for 2 × 5 minutes, subsequent Wash out the xylene with 100% vol / vol isopropanol and then gradually Replace the isopropanol with water in one pass a descending series of isopropanol in water dilutions. The last stage the dilution series, namely the 100% aqua bidest is preferably with a surfactant, for example 0.1% Tween 20 added.

After that the tissue section is dried on the nitrocellulose membrane and can then either directly subjected to the detection procedure or until further treatment be stored.

The abbreviation M is used below for molar (a solution is 1 M if it contains 1 mol of dissolved substance per liter; 10 ^-3 = millimolar (mM)).

For the detection according to the invention, for example the scrapie isoform of the 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 section lying on it are first soaked in TBST (10 mM TrisHCl, pH 7.8; 100 mM NaCl; 0.05% Tween 20) and then for about 8 hours in a closed incubation chamber at approx 55 ° C on a pad made of absorbent material, for example one or more cellulose layers, which are incubated with a solution of commercially available Proteinase K (eg from 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, for example by dropping or overlaying. The amount of proteinase K solution intended for wetting is chosen so small that the liquid added, for example the cellulose layer (s), can be almost completely absorbed by the substrate and no liquid level is formed which 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 arranged and is not covered by the solution - except for scheduled wetting. 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.

in the The nitrocellulose membrane is connected to the incubation with Proteinase K solution (including the largely digested tissue section) 3 × 5 minutes initially in TBST washed and then for 10-20 Minutes at room temperature in 4 M guanidinium (iso) thiocyanate solution (GdnSCN) incubated in 10 mM TrisHCl pH 7.8 to the proteins in the nitrocellulose membrane to denature. Then the GdnSCN with TBST 3 × 5 minutes washed out and the nitrocellulose membrane in 0.2% casein in TBST as a block reagent (for blocking, i.e. occupying non-specific Binding sites for the primary antibody subsequently used) for about 30 minutes. Then will the nitrocellulose membrane with a primary antibody against the prion protein is directed, for example the commercially available antibody BF4 (Company dako) for 1-12 hours incubated. For this will the antibody with blocking reagent to the antibody-specific concentration, those commonly used in the prior art for Western blot immune reactions is used, diluted.

at Incubation times of more than 3 hours are carried out at 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 incubated with the secondary antibody in the dilution specified by the manufacturer with blocking reagent for 1-12 hours under the same incubation conditions as for the primary antibody. In the case of a monoclonal primary antibody from a mouse, a goat anti-mouse antibody coupled with the enzyme "alkaline phosphatase" (for example from Dako, D0486), or in the case of a polyclonal primary antibody from a rabbit, is used as the secondary antibody Zie ge-anti-rabbit antibody. In any case, the secondary antibodies should be purified to avoid unwanted non-specific background reactions.

To visualize the immune reaction, the nitrocellulose membrane is subjected to a color reaction. For this purpose, the nitrocellulose membrane is first thoroughly washed with TBST for 5 × 10 minutes. 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 in the dark for 7-75 minutes (depending on the primary antibody) subjected. 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 The detection reaction is evaluated under a stereo magnifier 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 corresponding enzymes instead of Proteinase K. In practice, digestion with protease (1mg / ml; e.g. from Sigma) or double digestion with protease (1mg / ml) and Proteinase K (250μg / ml) for tissue sections containing connective tissue (tissue outside the central one) has also proven particularly effective nervous system).

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incubation

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casing

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ground

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absorbent material

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housing wall

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Digestive enzyme solution

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Nitrocellulose membrane

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surface

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tissue section

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liquid level

Claims (7)

Method for the detection of denatured proteins in formalin-fixed and paraffin-embedded tissue sections, characterized in that the proteins from the tissue sections on 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 according to type and order the following process steps are transferred and detected: 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 ci) the protease solution at a distance from the The top of the membrane and the tissue section is arranged 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 that is moistened with the protease solution, and wherein cii) the tissue section is periodic is drizzled or overlaid with protease solution, d) washing the membrane, and e) incubating the membrane with protein denaturing agent. A method according to claim 1, characterized in that the membrane is a nitrocellulose membrane or other suitable Membrane is. Membrane with top and bottom with Proteins arranged on the upper side are obtained from a tissue section by a method according to claim 1 or 2, characterized in that the topographic arrangement of the proteins on the membrane of the original corresponds to the topographical arrangement in the tissue section. Device for carrying out a method according to claim 1 or 2, characterized in that it comprises a housing ( 4 ) with floor ( 8th ), Wall and without or with a removable cover that has on the bottom ( 8th ) a layer ( 10 ) made of absorbent material ( 12 ) is arranged, the external dimensions of which are selected so that the layer ( 10 ) with an almost central arrangement on the floor ( 8th ) a distance from the wall ( 14 ) and their surface facing away from the case bottom ( 18 ) as a support for a membrane ( 20 ) and that on the floor ( 8th ) a protease solution ( 16 ) at a distance from the surface ( 18 ) the membrane ( 20 ) is arranged which the layer ( 10 ) wet or from the absorbent material of the layer ( 10 ) is included. Device according to claim 4, characterized in that the housing ( 4 ) is trough-shaped. Apparatus according to claim 4 or 5, characterized characterized that the layer ( 10 ) is one layer. Device according to claim 4 or 5, characterized in that the layer ( 10 ) consists of several layers, whereby the individual layers consist of the same absorbent material or of different absorbent materials.