CN117233393A - Double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors - Google Patents
Double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors Download PDFInfo
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Abstract
The invention belongs to the technical field of immunohistochemistry, and particularly relates to a double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors. The invention aims to solve the technical problem that the epithelial tumors of benign and malignant bile ducts are difficult to judge rapidly and accurately. The technical scheme for solving the technical problem is to provide a double immunohistochemical staining kit, which comprises a primary antibody reagent, a secondary antibody reagent and a staining agent; the primary antibody comprises an EZH2 antibody and a CK7 antibody, and the secondary antibody comprises an EZH2 secondary antibody and a CK7 secondary antibody. The technical scheme of the invention has better application prospect in identifying benign and malignant bile duct epithelial tumor cell lines, parting and judging the malignancy degree, and improving the diagnosis sensitivity and accuracy.
Description
Technical Field
The invention belongs to the technical field of immunohistochemistry, and particularly relates to a double-immunohistochemical staining kit and application thereof in identifying benign and malignant bile duct epithelial tumors.
Background
Immunohistochemistry (IHC), also known as immunocytochemistry, abbreviated immunohistochemistry, is the most important platform technology for pathological diagnosis, differential diagnosis, screening and identification of tumor specific molecular targets. Conventional immunohistochemistry is to determine intracellular antigens (polypeptides and proteins) of tissues by developing color developing agents (luciferin, enzymes, metal ions, isotopes) of labeled antibodies through chemical reaction by applying the basic principle of immunology, namely antigen-antibody reaction, namely the principle of specific binding of antigen and antibody, and to perform research on localization, qualitative and relative quantification. Generally, a section is observed for staining of an antigen. Multicolor immunohistochemistry can observe the staining results of a plurality of antigens on one slice, and the relation between different antigens is directly compared, so that the observation of tissue and tumor micro infiltration is facilitated. At present, the multicolor immunohistochemical technology is already applied to pathological diagnosis of various diseases such as prostate, mammary gland, digestive tract and the like, and has the technical difficulty of finding two or more proper markers to use primary antibodies and secondary antibodies to jointly mark on the same tissue section so as to achieve the effects of clear color contrast and accurate positioning.
Intrahepatic cholangiocarcinoma (ICC) accounts for about 10% -20% of primary liver cancer, 6% -20% of cholangiocarcinoma, and is the second most common liver malignancy with incidence inferior to hepatocellular carcinoma (HCC). ICC has highly malignant biological behaviors, and the survival rate of ICC patients after operation is generally between 30% and 50% at present. Many causative factors are known to be intimately involved in the development of ICC, including clonorchis sinensis, intrahepatic bile duct stones, chronic cholangitis, HBV/HCV infection, primary sclerosing cholangitis, parasitic infection, bile duct deformity (choledocholithiasis and Caroli disease), cirrhosis, congenital liver fibrosis, and the like. For those patients with underlying diseases of chronic liver injury, including cholestatic liver injury, intrahepatic and/or extrahepatic biliary stenosis, and other biliary abnormalities, reactive proliferation of the fine bile duct may be caused by repeated stimulation of inflammation, and may be further malignant, secondary ICC or HCC.
The common bile duct reaction is a reactive lesion in the limiting plate portal area, and comprises common bile duct hyperplasia, interstitial fibrosis and inflammatory cell infiltration, and is found in advanced chronic liver diseases (such as chronic viral hepatitis) and also around liver tumors such as HCC and ICC in advanced chronic liver diseases. When cellular atypical properties of ICC are not apparent, the histological and biliary reactions of ICC are very similar, and ICC often behaves as an infiltration alternative growth pattern, sometimes making it difficult to identify ICC and excessive biliary hyperplasia.
Bile duct adenoma is a benign tumor or neoplastic lesion, about 82.9% single shot, a few of which can be multiple shot. The size is usually <1cm, and the capsule is positioned under the liver capsule, or the deep part of liver parenchyma can exist, and the capsule is generally white or off-white, round or oval, harder in texture, clear in boundary and free of a real capsule. The small bile duct with consistent size is gathered under the lens, the lumen is narrow, the bending or solid strip is in a rope shape, and the small bile duct does not contain concentrated bile, is similar to bile duct reaction, and can be the early lesion of ICC. The biological properties of bile duct adenomas have not been fully elucidated so far, and differential diagnosis of bile duct adenomas from ICC is sometimes extremely difficult.
New WHO tumors (2019 edition) digestive system tumor classification criteria (WHO Classification of Tumours Editorial Board: digestive System Tumours, 5) th edition, 2019, vol.1; ICC in Http:// pubicions. Iarc. Fr/529), particularly adenocarcinomas originating from the common bile duct or the Hering duct, may sometimes resemble hyperplasia of the bile duct or reactive bile duct histologically. CLC cancer cells are in a small cube shape, have an increased nuclear/plasma ratio, are nuclear oval, pale in cytoplasm, lack mucus, are small in atypical, are in a highly differentiated state, are organized histologically into more regular angled small ducts, fissures or strips, and are characterized by an open branched or "deer horn" arrangement within the transparent denatured collagen fibrous matrix. For hyperdifferentiated CLC it is sometimes desirable to identify bile duct hamartoma and bile duct adenoma.
Diagnosis of hyperreactivity due to inflammation, bile duct hamartoma, bile duct lamina deformity, bile duct adenoma, or hyperdifferentiated ICC can sometimes be very challenging. While biliary epithelium, sometimes present simultaneously in liver malignancies, is truly neoplastic or reactive, is mixed HCC-ICC or HCC with biliary response, identification of these is also critical for accurate typing and correct diagnosis of malignancy. Thus, there is a great need in the art for other tools such as immunohistochemical staining to improve diagnostic sensitivity and accuracy.
Drosophila flyzesteThe human homolog 2 (Enhancer of zeste homolog, EZH 2) of the gene enhancer has the regulation and control functions of DNA methyl transfer and histone deacetylase, and can be involved in regulating and controlling the cancer progression, the multipotency of stem cells and other vital activities. It was found that its expression in ICC was significantly elevated. Sasaki et al found that EZH2 was highly expressed in all CLCs and not in cholangioma and reactive little bile duct hyperplasia (Am JSYRG Pathol 2014; 38:364). Therefore, immunohistochemical staining of EZH2 can be used to identify ICC (positive), excessive biliary reaction hyperplasia, bile duct hamartoma, bile duct plate deformity, bile duct adenoma (negative). CK7 (cytokeratin 7) is a member of the keratin gene family and can be used to delineate the biliary epithelium (Pathol Res practice 2003; 199:65).
Currently, in daily pathological diagnosis, various immunohistochemical tests are often required. However, due to technical limitations, the common immunohistochemical method is to label a protein with a color (typically brown) on 1 pathological tissue slide. When analyzing multiple protein indicators, it is necessary to label each of the multiple different slides with a corresponding antibody. The disadvantages of such a single-label immunohistochemical staining method are as follows: (1) each marker requires a tissue slice, and the specimen is wasted; (2) whether two immunohistochemical indexes respectively stained on two glass slides are simultaneously expressed on the same group of cells or not requires repeated comparison of HE and immunohistochemical sections by experience and conjecture, and has great difficulty in reading the sheets. Inexperienced physicians often misinterpret the bile duct epithelium that is mildly dysplastic in malignant tumors as malignant, resulting in misdiagnosis.
Disclosure of Invention
The invention aims to solve the technical problem that the epithelial tumors of benign and malignant bile ducts are difficult to judge rapidly and accurately.
The invention firstly provides a double immunohistochemical staining kit. The kit comprises a primary antibody reagent, a secondary antibody reagent and a staining agent; the primary antibody comprises an EZH2 antibody and a CK7 antibody; the secondary antibody reagent comprises a combined secondary antibody (HRP-labeled anti-mouse IgG/HRP+rabbit anti-IgG/AP) or a combined secondary antibody (HRP-labeled anti-mouse IgG/AP+rabbit anti-IgG/HRP), and the secondary antibody reagent comprises an EZH2 secondary antibody and a CK7 secondary antibody.
Wherein the EZH2 antibody and CK7 antibody are derived from animals of two different species. Further, the animals of the two different species are mice or rabbits, respectively.
Wherein the staining agent is a peroxidase HRP-mediated staining agent and an alkaline phosphatase AP-mediated staining agent.
Wherein the sources of antibodies to the same antigen in the primary and secondary antibodies are matched. Further, the EZH2 antibody is derived from a mouse or a rabbit, the CK7 antibody is derived from a rabbit or a mouse, and the EZH2 antibody and the CK7 antibody are derived from different animals.
Further, the secondary antibody contains horseradish peroxidase (HRP) -labeled secondary antibody polymer matched with the species source of the EZH2 or CK7 antibody in the primary antibody; the secondary antibody reagent contains an Alkaline Phosphatase (AP) -labeled secondary antibody polymer that matches the species source of the EZH2 or CK7 antibodies in the primary antibody reagent; the EZH2 secondary antibody and the CK7 secondary antibody in the secondary antibody reagent are marked differently.
For example, the secondary antibody reagent comprises a combined secondary antibody (HRP-labeled anti-mouse IgG/HRP+rabbit anti-IgG/AP) or a combined secondary antibody (HRP-labeled anti-mouse IgG/AP+rabbit anti-IgG/HRP).
The double immunohistochemical staining kit further comprises a staining agent, wherein the staining agent comprises horseradish peroxidase substrate staining solution and alkaline phosphatase substrate staining solution.
Wherein the horseradish peroxidase substrate staining solution comprises 3, 3-diaminobenzidine hydrochloride (DAB); alternatively, the alkaline phosphatase substrate staining solution comprises a Red alkaline phosphatase substrate (specifically, fast Red a and Fast Red B).
Further, the dye further comprises a counterstain dye solution, and the color development of the counterstain dye solution does not cover the color development of the horseradish peroxidase substrate dye solution and the alkaline phosphatase substrate dye solution.
Wherein, the double-dyeing immunohistochemical kit can also contain an antigen retrieval agent and/or an endogenous catalase blocking reagent.
Further, the double immunohistochemical staining kit is used for staining tissue sections. Further, the tissue sections are paraffin tissue sections and frozen tissue sections.
It will be appreciated that the various reagents in the above-described kit are individually packaged.
The invention further provides application of the double immunohistochemical staining kit in preparation of a reagent for identifying benign and malignant bile duct epithelial tumors.
The invention also provides a method for double immunohistochemical staining of liver tissue sections based on the scheme. The method is characterized by comprising the following three operation modes:
A：
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Tissue sections were incubated with primary antibody: adopting a cocktail primary antibody, wherein the cocktail primary antibody comprises a murine EZH2 primary antibody and a rabbit CK7 primary antibody, or comprises a rabbit EZH2 primary antibody and a murine CK7 primary antibody;
(3) Secondary antibody incubation and color reaction: incubation with horseradish peroxidase (HRP) labeled secondary antibodies matched to the species source of EZH2 or CK7 antibodies, mediated 3, 3-diaminobenzidine hydrochloride (DAB) reaction, resulting in a brown or tan precipitate that is insoluble in water and ethanol; then, performing incubation-mediated red substrate chromogenic system staining by using Alkaline Phosphatase (AP) labeled secondary antibodies matched with the residual CK7 or EZH2 antibody species sources;
(4) Slicing and counterstaining; and (5) storing for standby.
Different results can be presented according to the enzyme-labeled secondary antibodies:
HRP-labeled secondary antibody binds to EZH 2/AP-labeled secondary antibody binds to CK7: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
HRP-labeled secondary antibody binds to CK 7/AP-labeled secondary antibody binds to EZH2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
Or B:
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Tissue sections were incubated with primary antibody: adopting a cocktail primary antibody, wherein the cocktail primary antibody comprises a murine EZH2 primary antibody and a rabbit CK7 primary antibody, or comprises a rabbit EZH2 primary antibody and a murine CK7 primary antibody;
(3) Secondary antibody incubation and color reaction: adopting cocktail secondary antibody, and simultaneously mediating enzymatic chromogenic reaction; the cocktail secondary antibody comprises horseradish peroxidase (HRP) labeled secondary antibody and Alkaline Phosphatase (AP) labeled secondary antibody respectively aiming at EZH2 or CK7, and is matched with the species source of the corresponding primary antibody; HRP mediates the formation of a brown or tan precipitate, AP mediates the formation of a red precipitate;
(4) Slicing and counterstaining; and (5) storing for standby.
Different results can be presented according to the enzyme-labeled secondary antibodies:
HRP-labeled secondary antibody binds to EZH 2/AP-labeled secondary antibody binds to CK7: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
HRP-labeled secondary antibody binds to CK 7/AP-labeled secondary antibody binds to EZH2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
Or C:
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Incubation with a murine or rabbit origin EZH2 or CK7 primary antibody, with a horseradish peroxidase (HRP) -labeled secondary antibody matched to the primary antibody species origin, mediates the 3, 3-diaminobenzidine hydrochloride (DAB) reaction, yielding a water-and ethanol-insoluble tan or brown precipitate;
(3) Secondary antibody incubation and color reaction: then incubating with a primary antibody of CK7 or EZH2 of murine or rabbit origin, which is different from the antigen from which the primary antibody was derived and against; after incubation, incubation with Alkaline Phosphatase (AP) -labeled secondary antibodies of the same species origin as the primary antibodies, mediated formation of red precipitates based on AP catalysis;
(4) Counterstaining; and (5) storing for standby.
Presenting different results according to the primary antibody selection order:
step one selects EZH 2/step two selects CK7: EZH2 brown nuclear staining and CK7 red cytoplasmic staining;
step one, CK 7/step two, EZH2: CK7 brown cytoplasmic staining and EZH2 red nuclear staining.
The double-staining slice prepared by the method can be used for microscopic observation to identify benign and malignant bile duct epithelial tumors.
The invention has the beneficial effects that: according to the technical scheme, a double immunohistochemical technology is utilized, and aiming at two related antigen protein indexes CK7 and EZH2, the reactive bile duct and cancerous bile duct epithelium can be simultaneously sketched on one slice, and the relation between the reactive bile duct and the cancerous bile duct epithelium can be observed, so that the identification of tumors and non-tumor bile ducts can be facilitated, and the correct pathological diagnosis is facilitated. By utilizing the technical scheme of the invention, whether two simultaneously marked antigen indexes are simultaneously expressed in the same group of cells is obtained, and the observation result under a microscope is directly used for judging, so that the judgment is not carried out by experience or speculation, the difficulty in film reading is reduced, the working efficiency and the judgment accuracy are improved, and the effect of 1+1>2 is achieved. On the other hand, as one glass slide can detect two protein indexes simultaneously, the use amount of tissue sections is reduced, and the method has good technical effects. Clinical experiments prove that the method has a good application prospect in identifying the benign and malignant bile duct epithelial tumor cell line, parting and judging the malignancy degree, and improving the diagnosis sensitivity and accuracy.
Drawings
Fig. 1, case 1 liver packet HE low-magnification photographs (4×).
Fig. 2, case 1 liver packet HE middle-magnification photographs (10×).
Fig. 3, case 1 liver block double-stained immunohistochemical low-magnification photographs (4×).
Fig. 4, case 1 liver block double-stained immunohistochemical medium-power photographs (10×).
Fig. 5, case 2 liver occupation HE middle-magnification photographs (10×).
FIG. 6, case 2 liver occupation space single-stained CK7 immunohistochemical medium-power photographs (10X).
Fig. 7, case 2 liver occupation single-stained EZH2 immunohistochemical medium-fold photographs (10×).
Fig. 8, case 2 liver occupancy double staining immunohistochemical medium-high power photographs (20×).
Fig. 9, case 3 liver packet HE low-magnification photographs (4×).
Fig. 10, case 3, liver packet HE middle-magnification photographs (10×).
Fig. 11, case 3 liver block double-stained immunohistochemical low-magnification photographs (4×).
Fig. 12, case 3 liver block double-stained immunohistochemical medium-high power photographs (20×).
Detailed Description
In the diagnosis of hematological disorders, it is known in the art to detect whether a population of cells has co-expression of a protein marker of interest or the absence of certain markers by flow cytometry techniques from fresh samples. However, in solid tumors, firstly fresh samples are not easy to obtain, and secondly, not all immunohistochemical protein antibodies are marked with flow cytometry antibodies, so that accurate diagnosis still depends on tissue sections. Thus, if benign and malignant components of bile duct epithelium can be identified on a tissue slice at the same time, the method has great practical significance for the field and is greatly helpful for first-line clinical work.
The invention creatively develops a double immunohistochemical staining technique aiming at two different markers EZH2 and CK7, discovers that EZH2 and CK7 are selected as antigen protein indexes to carry out antigen immunostaining with different colors, can simultaneously identify benign and malignant components of bile duct epithelium on one slice, deepens further understanding of ICC and reactive bile duct, and is beneficial to the accurate diagnosis of tumors.
The invention relates to a double immunohistochemical staining kit. The kit comprises a primary antibody reagent, a secondary antibody reagent and a staining agent; the primary antibody comprises an EZH2 antibody and a CK7 antibody; the secondary antibody reagent comprises a combined secondary antibody (HRP-labeled anti-mouse IgG/HRP+rabbit anti-IgG/AP), wherein the mixed solution of the secondary antibody can make the labeled part of the primary antibody of the mouse source and DAB develop into brown and the labeled part of the primary antibody of the rabbit source and AP develop into red), or a combined secondary antibody (HRP-labeled anti-mouse IgG/AP+rabbit anti-IgG/HRP), and the mixed solution of the secondary antibody can make the labeled part of the primary antibody of the mouse source and AP develop into red and the labeled part of the primary antibody of the rabbit source and DAB develop into brown. In general, the EZH2 antibody and CK7 antibody are preferably derived from animals of two different species. For example, animals of two different species are mice or rabbits, respectively. The coloring agent can be various coloring agents commonly used in the art. Such as the use of the widely used peroxidase HRP-horseradish mediated stain and Alkaline Phosphatase (AP) -mediated stain. It is also a task to optimize the specific dyeing operations to obtain a more contrasting, clearer and clearer sheet.
The method can respectively or simultaneously perform double color development on the same tissue slice, and the dyeing contrast is vivid and visual. Through multi-factor (different technicians, different tissues and the like) comparison and verification, antigens are not lost, antibodies are not interfered with each other, and expert interpretation results are completely consistent. Has good popularization value.
The following examples are provided to further illustrate the technical aspects of the present invention in detail, but are not intended to limit the scope of the present invention.
The names and the purchased manufacturers of the main reagents specifically used in the embodiment of the invention are as follows:
CK7 primary antibody, clone number: UMAB161, murine mab, ready-to-use, sequoyitol bridge;
EZH2 primary antibody, clone number: SP129, rabbit monoclonal antibody, ready-to-use, gene technology;
ultraView Universal DAB Detection Kit Roche (Roche), REF 760-500, which is an HRP-labeled secondary antibody kit used by the Ventana staining platform, the kit comprising DAB color development liquid.
ultraView Universal Alkaline Phosphatase Red Detection Kit, roche, REF 760-501, which is an AP-labeled secondary antibody kit used by the Ventana staining platform, the kit comprises an AP red color solution.
Roche Ventana BenchMark immunohistochemical apparatus.
EXAMPLE 1 preparation of post-operative double-stained immunohistochemical sections Using the technique of the present invention
Case 1 to be diagnosed in this example is a 66 year old female patient, due to "skin and scleral yellow 3 + Month, examination found liver occupation 1 + Month "admitted; post-operative pathology see fig. 1 and 2.
Fig. 1 is HE low (4×): under the liver capsule there is more hyperplastic bile duct epithelium, which is arranged in the form of irregular solid streak, deer horn or branch, loose interstitial, unobvious fibrosis and unclear boundary. These lumens were either reactive or neoplastic at all, and differential diagnosis included: bile duct adenoma, intrahepatic bile duct cancer (fine bile duct cancer).
Fig. 2 is a middle-multiple of HE (10×): bile duct cells are small cubes, similar to bile duct epithelial cells, are consistent in size, have few partial cytoplasm, increase nuclear plasma ratio, have oval nucleuses, are rare in nuclear division and are difficult to diagnose.
To enable clear diagnosis, CK7 and EZH2 double-stained immunohistochemistry was performed on patient tissues.
The specific preparation steps are as follows:
1. preparing paraffin sections of 4 mu m, and baking the sections for 30 minutes at 65 ℃;
2. dewaxing and antigen retrieval using Roche Ventana BenchMark;
3. dripping EZH2 primary antibody, incubating ultraView Universal DAB Detection Kit secondary antibody after 36 minutes, and completing DAB color development;
4. dripping CK7 primary antibody, incubating ultraView Universal Alkaline Phosphatase Red Detection Kit secondary antibody after 64 minutes, and finishing AP red color development;
5. the blue-turning liquid turns blue after 4 minutes of hematoxylin counterstain;
6. oven drying at 65deg.C for 3 hr after washing with tap water, and observing under a mirror after sealing the neutral resin.
And 2-5 dyeing steps are finished in Roche Ventana BenchMark dyeing platform operation.
The results after double dyeing are shown in fig. 3 and 4.
Fig. 3 is double-stained immunohistochemical low power (4×): the surrounding bile duct epithelium and the right bile duct epithelium ck7+ezh2- (blue arrow indicates, showing red, only cell membrane stained red), while the central and upper left corners are red+brown, i.e. ck7+ezh2+ (reddish brown, red cell membrane+brown nucleus), suggesting that the bile duct in the central and upper left corners is bile duct adenocarcinoma. Whereas the peripheral and upper right corners are reactive/residual bile duct epithelium, not tumorous.
Fig. 4 is double-stained immunohistochemistry medium (10×): blue arrows are reactive bile duct epithelium, red arrows are bile duct cancer, and the contrast is clear, so that the diagnosis is facilitated.
From various staining charts of the case, the cell morphology after double staining is clear, the cell nucleus, cell membrane and cytoplasm are clear in color, positive positioning is accurate, signal expression is clear, and the double staining method can be applied to differential diagnosis of bile duct adenocarcinoma and reactive/residual bile duct epithelium.
Example 2 preparation of post-operative double-stained immunohistochemical sections Using the technique of the present invention
Case 2 to be diagnosed in this example is a 60 year old male patient admitted as "physical examination finds liver to occupy 20 days";
post-operative pathology is seen in fig. 5-7.
Fig. 5 is a middle-multiple of HE (10×): the arrangement of spindle-shaped cells is in the form of bundles and sheets, and the abnormal shape is obvious. The cells have certain allotype, and the lumen-like structure or the strip-like structure formed by the scattered single-layer cubic epithelium is hidden and visible in the cell. These invisible lumens are ultimately reactive or neoplastic, and are highly controversial by different doctors. Diagnosis is considered to be spindle cell malignancy. The differential diagnosis includes: sarcoidosis hepatocellular carcinoma, intrahepatic bile duct carcinoma + sarcoidosis carcinoma, sarcoma, etc.
FIG. 6 is a single-stained CK7 immunohistochemistry at multiple (10X): CK7 shows bile duct epithelium in tumor bodies, seen adenoid arrangement, few irregularities, seen deer horn and irregularities, but it is not clear whether these bile ducts are reactive or neoplastic at all.
Figure 7 is a single-stained EZH2 immunohistochemical medium-fold (10×): the high expression of spindle cells in the tumor body is shown by EZH2, the bile duct epithelium of the approximately same part is found by repeatedly comparing HE sections and immunohistochemistry of CK7, and the bile duct epithelium is found to be negative expression (indicated by arrows) through careful comparison and identification.
To enable clear diagnosis, the present technology was used to double-stained immunohistochemical for CK7 and EZH2 on tissues.
The specific steps of dyeing into tablets are as follows:
1. preparing paraffin sections of 4 mu m, and baking the sections for 30 minutes at 65 ℃;
2. dewaxing and antigen retrieval using Roche Ventana BenchMark;
3. dripping EZH2 primary antibody, incubating ultraView Universal DAB Detection Kit secondary antibody after 36 minutes, and completing DAB color development;
4. dripping CK7 primary antibody, incubating ultraView Universal Alkaline Phosphatase Red Detection Kit secondary antibody after 64 minutes, and finishing AP red color development;
5. the blue-turning liquid turns blue after 4 minutes of hematoxylin counterstain;
6. oven drying at 65deg.C for 3 hr after washing with tap water, and observing under a mirror after sealing the neutral resin.
And 2-5 dyeing steps are finished in Roche Ventana BenchMark dyeing platform operation.
The results after double dyeing are shown in FIG. 8. FIG. 8 is a high-power photograph (20X) of double staining immunohistochemistry, and it can be seen that a single layer of cubic epithelium surrounds the formed lumen-like structure CK7+EZH2- (red, cell membrane), and spindle cells CK7-EZH2+ (brown, cell nucleus) on one slice, and the color contrast is vivid, and repeated contrast viewing is not required. This suggests that bile duct epithelium present in spindle cell tumors is reactive and/or residual bile duct epithelium, not a malignant component. I.e. not ICC, nor hybrid HCC-ICC, contributes to a correct pathological diagnosis. The cell morphology after double-dyeing is clear, the cell nucleus, cell membrane and cell plasma are clear in color, the positive positioning is accurate, the signal expression is clear, and the method can be applied to differential diagnosis of bile duct adenocarcinoma and reactive/residual bile duct epithelium.
EXAMPLE 3 preparation of post-operative double-stained immunohistochemical sections Using the technique of the present invention
Case 3 to be diagnosed in this example is a 39 year old female patient, due to "intermittent abdominal distension and pain 10 + Years, the diagnosis of antral adenocarcinoma is carried out for 1 month; the left hepatic margin surface was found to be 1 gray-white nodule of about 0.3cm in diameter, which was taken to give pathology during surgery, and it was desired to determine whether it was the primary metastatic adenocarcinoma of the stomach. Post-operative paraffin section pathology see fig. 9 and 10.
Fig. 9 is HE low (4×): the isolated nodules are seen under the liver capsule, which is the bile duct epithelium with more hyperplasia, the size is more consistent, the lumen is narrow, the curve shape or the irregular solid strip rope shape is formed, the cystic cavity or the bile duct is not expanded, the interstitial is visible to be infiltrated by lymphocyte, and the boundary is not clear. These lumens were either reactive or neoplastic at all, and differential diagnosis included: bile duct adenoma, intrahepatic bile duct cancer (fine bile duct cancer), metastatic adenocarcinomas.
Fig. 10 is a middle HE multiple (10×): it can be seen that bile duct cells are cubic or short columnar, have no obvious abnormal shape, are rich in cytoplasm, have small round cell nuclei, have uniform size, have no obvious mitotic image, and are difficult to diagnose.
To enable clear diagnosis, CK7 and EZH2 double-stained immunohistochemistry was performed on patient tissues. Double-dye immunohistochemical staining was also performed in this example following the staining protocol of the above example. The results after double dyeing are shown in fig. 11 and 12.
FIG. 11 is a double staining immunohistochemical low magnification photograph (10X), and it can be seen that a single layer of cubic epithelium surrounds the formed lumen-like structure CK7+EZH2- (red, cell membrane) and shows normal bile duct epithelium in the portal area of peripheral liver tissue, whereas no brown bile duct epithelium of CK7+EZH2+ (brown, cell nucleus) is seen, and the color is vivid. Indicating that these bile duct epithelia are benign lesions, not malignant components. And is helpful for correct pathological diagnosis.
FIG. 12 is a high-power photograph (20X) of double-staining immunohistochemistry, wherein a single-layer cubic epithelium surrounds a formed lumen-like structure CK7+EZH2- (red, cell membrane), CK7-EZH2+ (brown, cell nucleus) to form reactive lymphocytes, and the reactive lymphocytes can also be used as a positive control, and the reactive lymphocytes and the positive control can be displayed on a slice, so that the color contrast is clear, the cell morphology after double-staining is clear, the cell nucleus, cell membrane and cell plasma are clear in color, the positive positioning is accurate, and the signal expression is clear, and can be applied to the differential diagnosis of cholangiocarcinoma, reactive/residual cholangiocarcinoma and metastatic adenocarcinomas.
Claims (10)
1. A double immunohistochemical staining kit, characterized in that: comprises a primary antibody reagent, a secondary antibody reagent and a staining agent; the primary antibody comprises an EZH2 primary antibody and a CK7 primary antibody, and the secondary antibody comprises an EZH2 secondary antibody and a CK7 secondary antibody; the EZH2 antibody and CK7 antibody were derived from mouse and rabbit, respectively.
2. The dual immunohistochemical staining kit of claim 1, wherein: the staining agent is horseradish peroxidase HRP mediated staining agent and alkaline phosphatase AP mediated staining agent.
3. The dual immunohistochemical staining kit of claim 1, wherein: the sources of antibodies to the same antigen in the primary antibody reagent and the secondary antibody reagent are matched; alternatively, the EZH2 antibody is derived from a mouse or rabbit, the CK7 antibody is derived from a rabbit or mouse, and the EZH2 antibody and the CK7 antibody are different.
4. The dual immunohistochemical staining kit of claim 1, wherein:
the secondary antibody reagent contains a horseradish peroxidase HRP-labeled secondary antibody polymer matched with the species source of the EZH2 or CK7 antibody in the primary antibody reagent;
the secondary antibody reagent contains alkaline phosphatase AP marked secondary antibody polymer matched with the EZH2 or CK7 antibody species source in the primary antibody reagent;
the EZH2 secondary antibody and the CK7 secondary antibody in the secondary antibody reagent are marked differently.
5. The dual immunohistochemical staining kit of claim 1, wherein: the dye also comprises a dye, wherein the dye comprises horseradish peroxidase substrate dye liquid and alkaline phosphatase substrate dye liquid.
6. The double immunohistochemical staining kit of claim 5, wherein: the horseradish peroxidase substrate staining solution comprises 3, 3-diaminobenzidine hydrochloride DAB; or the alkaline phosphatase substrate staining solution comprises a red alkaline phosphatase substrate.
7. The double immunohistochemical staining kit of claim 6, wherein: the dye further comprises counterstain dye liquor, and the color development of the counterstain dye liquor does not cover the color development of the horseradish peroxidase substrate dye liquor and the alkaline phosphatase substrate dye liquor.
8. The dual immunohistochemical staining kit of claim 1, wherein the dual immunohistochemical staining kit is used for staining tissue sections; the tissue slice is a paraffin tissue slice or a frozen human tissue slice.
9. Use of the double immunohistochemical staining kit according to any one of claims 1 to 8 in the preparation of a reagent for identifying benign and malignant bile duct epithelial tumors.
10. The method for double immunohistochemical staining of liver tissue sections is characterized by comprising the following steps:
A：
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Tissue sections were incubated with primary antibody: adopting a cocktail primary antibody, wherein the cocktail primary antibody comprises a murine EZH2 primary antibody and a rabbit CK7 primary antibody, or comprises a rabbit EZH2 primary antibody and a murine CK7 primary antibody;
(3) Secondary antibody incubation and color reaction: incubating with horseradish peroxidase (HRP) -labeled secondary antibody matched with the species source of EZH2 or CK7 antibody, and mediating DAB reaction of 3, 3-diaminobenzidine hydrochloride to generate brown yellow or brown precipitate insoluble in water and ethanol; then, performing incubation-mediated red substrate chromogenic system staining on the alkaline phosphatase AP labeled secondary antibody matched with the residual CK7 or EZH2 antibody species sources;
(4) Slicing and counterstaining; preserving for standby;
or B:
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Tissue sections were incubated with primary antibody: adopting a cocktail primary antibody, wherein the cocktail primary antibody comprises a murine EZH2 primary antibody and a rabbit CK7 primary antibody, or comprises a rabbit EZH2 primary antibody and a murine CK7 primary antibody;
(3) Secondary antibody incubation and color reaction: adopting cocktail secondary antibody, and simultaneously mediating enzymatic chromogenic reaction; the cocktail secondary antibody comprises a horseradish peroxidase HRP-labeled secondary antibody and an alkaline phosphatase AP-labeled secondary antibody which are respectively aimed at EZH2 or CK7, and is matched with the species source of the corresponding primary antibody; HRP mediates the formation of a brown or tan precipitate, AP mediates the formation of a red precipitate;
(4) Slicing and counterstaining; preserving for standby;
or C:
(1) Preparing a tissue section, and carrying out antigen retrieval and endogenous catalase blocking;
(2) Incubation with a murine or rabbit origin EZH2 or CK7 primary antibody, with a horseradish peroxidase HRP-labeled secondary antibody matched to the primary antibody species origin, mediates the 3, 3-diaminobenzidine hydrochloride DAB reaction, yielding a water and ethanol insoluble brown or tan precipitate;
(3) Secondary antibody incubation and color reaction: then incubating with a primary antibody of CK7 or EZH2 of murine or rabbit origin, which is different from the antigen from which the primary antibody was derived and against; after incubation, the secondary antibody is marked by alkaline phosphatase AP which is the same as the primary antibody in origin for incubation, and red sediment is mediated based on AP catalysis;
(4) Counterstaining; and (5) storing for standby.
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