An immunohistochemical double-staining three-marker kit for breast invasive carcinoma Ki-67 index interpretation

Abstract

The application provides an immunohistochemical double-staining three-marker kit for breast invasive carcinoma Ki-67 index interpretation, which comprises a mixed primary antibody; the mixed primary antibody comprises a mouse antibody and a rabbit antibody; the mixed primary antibody comprises a myoepithelial marker antibody, a glandular epithelial marker antibody and a Ki-67 antibody; the myoepithelial marker antibody and the glandular epithelial marker antibody are located in the cytoplasm and / or cell membrane. Different from the prior art, the above technical scheme adopts the mixed primary antibody of the myoepithelial marker antibody, the glandular epithelial marker antibody and the Ki-67 antibody, can simultaneously and synchronously mark on the same breast or breast cancer tissue section, and accurately and quickly distinguishes the invasive carcinoma and the carcinoma in situ and the interstitial and lymphocyte regions through the immunohistochemical multiplex staining mode. Combined with morphological characteristics, through the location and color difference, the invasive carcinoma region is obviously displayed, and the percentage of Ki-67 positive cells in the invasive carcinoma region is calculated.

Description

TECHNICAL FIELD

[0001] The application relates to the field of immunohistochemical technology, and particularly relates to an immunohistochemical double-staining three-marker kit for breast invasive carcinoma Ki-67 index interpretation. BACKGROUND

[0002] Ki-67 is a kind of cell cycle-related proliferating cell nuclear antigen, and is one of the most widely used cell proliferation markers. Studies have shown that Ki-67 positive expression can be used as an important indicator for judging the biological behavior of breast cancer, and the expression level is related to the stage of breast cancer and lymph node metastasis, and is also an important indicator for studying cell proliferation activity. Studies have shown that Ki-67 can guide the prognosis of breast cancer and guide the treatment of breast cancer, and Ki-67 has very important significance in clinical practice.

[0003] According to the 2021 CSCO Breast Cancer Diagnosis and Treatment Guidelines, Ki-67 detection should be performed on all breast invasive carcinoma cases, and the percentage of positive staining cells in the nucleus of cancer cells should be reported. Positive definition is any degree of brown staining of invasive carcinoma cell nucleus.

[0004] In 2021, the "Breast Cancer Ki-67 International Working Group Assessment Guidelines" recommend using the standardized "typewriter" visual assessment method for interpretation, and there should be a strict quality assessment and control system to ensure the effectiveness of the analysis. According to the latest evaluation standard, the working group clearly defines Ki-67>30% as high expression. In clinical practice, when facing HR-positive, HER-2-negative, T1N0 breast cancer patients, if Ki-67 is highly expressed, it is obviously unnecessary to perform gene detection to decide whether to perform chemotherapy.

[0005] The inventors found that the prior art has the following problems: In daily work, when H&E staining sections are combined with Ki-67 single staining for semi-quantitative interpretation, it is difficult to distinguish between invasive carcinoma and carcinoma in situ, and the stroma and lymphocytes (which can also be Ki-67 positive) may cause deviation in the percentage of Ki-67 positive tumor cells. The interpretation result of Ki-67 is strictly dependent on the clinical experience of clinical pathologists, and the result is all estimated, which is greatly influenced by the subjectivity of clinical pathologists. SUMMARY

[0006] In view of the above problems, the application provides an immunohistochemical double-staining three-marker kit for breast invasive carcinoma Ki-67 index interpretation, which comprises: a mixed primary antibody; the mixed primary antibody comprises mouse and rabbit antibodies; the mixed primary antibody comprises a myoepithelial marker antibody, a glandular epithelial marker antibody and a Ki-67 antibody; the myoepithelial marker antibody and the glandular epithelial marker antibody are located in the cytoplasm and / or cell membrane.

[0007] Different from the prior art, the technical scheme adopts mixed primary antibodies of the myoepithelial marker antibody, the glandular epithelial marker antibody and the Ki-67 antibody, can simultaneously and synchronously mark on the same breast or breast cancer tissue section, and accurately and quickly distinguishes the invasive carcinoma and the carcinoma in situ and the intercellular substance and the lymphocyte area through the immunohistochemical multiplex staining. Combined with the morphological characteristics, the invasive carcinoma area is obviously displayed through the positioning and color difference, and the percentage of the Ki-67 positive cells in the invasive carcinoma area is calculated.

[0008] In some embodiments, the myoepithelial marker antibody comprises CK5, Calponin and p63 / p40.

[0009] In some embodiments, the myoepithelial marker antibody is CK5.

[0010] In some embodiments, the glandular epithelial marker antibody comprises CK8 / 18, CK(pan).

[0011] In some embodiments, the glandular epithelial marker antibody is CK8 / 18.

[0012] In some embodiments, the myoepithelial marker antibody, the glandular epithelial marker antibody and the Ki-67 antibody adopt monoclonal antibodies.

[0013] In some embodiments, the glandular epithelial marker and the Ki-67 adopt different species of antibodies.

[0014] In some embodiments, the Ki-67 adopts a rabbit monoclonal antibody, the glandular epithelial marker antibody adopts a CK8 / 18 mouse monoclonal antibody, and the myoepithelial marker antibody adopts a CK5 rabbit monoclonal antibody.

[0015] In some embodiments, the kit further comprises mixed secondary antibodies and a color developing solution; the mixed secondary antibodies comprise HPR-goat anti-rabbit secondary antibodies and AP enzyme-labeled secondary antibodies, and the color developing solution comprises DAB color developing solution and AP-RED color developing solution.

[0016] In some embodiments, in the color developing step, the DAB color developing solution is used to develop the Ki-67 and the CK5, and the AP-RED color developing solution is used to develop the CK8 / 18.

[0017] The above invention content is only a summary of the technical scheme of the present application. In order to enable those skilled in the art to more clearly understand the technical scheme of the present application, and then can be implemented according to the content of the description and the drawings, and in order to let the above-mentioned purpose and other purposes, characteristics and advantages of the present application can be more easily understood, the following is described in combination with the specific embodiments of the present application and the drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] The accompanying drawings are only used to illustrate the principles, implementation manners, applications, characteristics and effects of the specific embodiments of the present application and cannot be considered as limitation to the present application.

[0019] In the drawings:

[0020] Figure 1 CK5 and p63 / p40 immunohistochemical staining results in breast myoepithelial cells;

[0021] Figure 2 CK5 and Calponin immunohistochemical staining results in breast myoepithelial cells;

[0022] Figure 3 CK8 / 18+CK5 and CK(pan)+CK5 immunohistochemical staining results comparison in breast cancer tissue chip;

[0023] Figure 4 Ki-67 results comparison using different developers DAB and AP-RED;

[0024] Figure 5 DAB and AP-RED different developing order results comparison;

[0025] Figure 6 Breast cancer immunohistochemical staining results using the double-staining three-marker kit described in Example 1. DETAILED DESCRIPTION

[0026] To explain the possible application scenarios, technical principles, specific implementable schemes, and the purposes and effects that can be achieved of the present application in detail, the following will be described in detail in combination with the specific examples listed and the accompanying drawings. The examples described in the present document are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, and cannot limit the protection scope of the present application.

[0027] In the present document, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, each technical feature mentioned in each embodiment can be combined in any manner to form a corresponding implementable technical solution.

[0028] Unless otherwise defined, the meanings of technical terms used in the present application are the same as commonly understood by one of ordinary skill in the art to which the present application belongs; the use of related terms in the present application is only for the purpose of describing specific embodiments and is not intended to limit the present application.

[0029] In the description of the present application, the phrase "and / or" is a description of the logical relationship between objects, which means that there can be three relationships, for example, A and / or B, which means that there are three cases: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally represents that the associated objects before and after are a "or" logical relationship.

[0030] In the present application, the terms such as "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual quantity, primary and secondary or order relationship between the entities or operations.

[0031] In the present application, the phrases "including", "containing", "having" or other similar expressions used in the sentence are intended to cover non-exclusive inclusion, and these phrases do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0032] In the present application, "greater than", "less than", "exceed" and other expressions are understood as not including the number; "above", "below", "within" and other expressions are understood as including the number. In addition, the meaning of "multiple" in the description of the embodiments of the present application is more than two (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly limited.

[0033] The present application provides a kit for interpreting breast invasive carcinoma Ki-67 index, which comprises: mixed primary antibody, the mixed primary antibody comprises mouse and rabbit antibodies; the mixed primary antibody comprises breast myoepithelial marker antibody, glandular epithelial marker antibody and Ki-67 antibody; the myoepithelial marker antibody and the glandular epithelial marker antibody are located in the cytoplasm and / or cell membrane.

[0034] The mixed primary antibody comprises mouse and rabbit antibodies. This design cooperates with different enzyme-labeled secondary antibody developing systems, so that mouse and rabbit antibodies can show different colors after immunohistochemical staining, and different colors can be used to label specific tissues or cells, which is convenient for pathologists to read and judge.

[0035] Due to the different types of breast, the myoepithelial cells constitute the morphology of the gland will be as follows: differences, so the applicant uses myoepithelial marker antibody as one of the primary antibodies.

[0036] Tissue type Myoepithelial cells form the shape of the periphery of the gland Normal breast / benign epithelial disease Intact Breast ductal carcinoma in situ Intact or not intact Breast invasive carcinoma Missing

[0037] And considering that Ki-67 will also be positive in the stroma and lymphocytes of non-tumor cells, in order to avoid adding Ki-67 in the stroma and lymphocytes to the statistics, the applicant also uses a highly specific gland epithelial marker that can specifically stain breast tissue but not stroma and lymphocytes. In this way, whether it is breast tissue can be determined according to the staining of the gland epithelial marker, and false counting of Ki-67 can be avoided.

[0038] At the same time, since Ki-67 is located in the nucleus, in order to facilitate differentiation, the localization of the myoepithelial marker antibody and the gland epithelial marker antibody is cytoplasmic or / and cell membrane.

[0039] The applicant found in the research that the antibody localized on the cell membrane occasionally had a missing situation in tumor cell staining, which may be due to the dissolution and loss of the cell membrane during the proliferation of cancer cells. Therefore, the localization of the myoepithelial marker antibody and the gland epithelial marker antibody is preferably cytoplasmic.

[0040] Unlike the prior art, the above technical solution uses a mixed primary antibody of myoepithelial marker antibody, gland epithelial marker antibody and Ki-67 antibody, which can simultaneously and synchronously label the same breast or breast cancer tissue section, accurately and quickly distinguish invasive carcinoma and carcinoma in situ, and the interstitial and lymphocyte regions through immunohistochemical multiplex staining. By distinguishing the location and color, the invasive carcinoma region is clearly displayed, and the percentage of Ki-67 positive cells in the invasive carcinoma region is easily calculated.

[0041] In some embodiments, the myoepithelial marker includes: CK5, Calponin and p63 / p40, all of which are common cells in myoepithelium. Preferably, CK5.

[0042] During the research and development process, the applicant respectively used CK5, Calponin and p63 / p40 for immunohistochemical single staining of breast tissue, and the results showed that P63 / p40 had poor staining continuity in breast myoepithelial tissue. In addition to staining breast myoepithelium, Calponin also stains smooth muscle and fibrocytes, which makes it difficult to distinguish between carcinoma in situ, microinvasive carcinoma and invasive carcinoma. CK5 has continuity in immunohistochemical staining of breast tissue myoepithelium, stable recognition, and does not have non-specific staining on other tissues.

[0043] Figure 1 The immunohistochemical staining results of CK5 and p63 / p40 in a case of mammary myoepithelial cells are shown in Figure 2. Figure 1 We can clearly see that p63 / p40 did not stain the epithelial cells, while CK5 stained the entire myoepithelial cells.

[0044] Figure 2 The immunohistochemical staining results of CK5 and Calponin in another case of mammary myoepithelial cells are shown in Figure 2. Figure 2 We can clearly see that the staining intensity of Calponin is lower than that of CK5, and it not only stains myoepithelial cells, but also fibroblasts and connective tissue; it cannot specifically mark myoepithelial cells.

[0045] In some embodiments, the glandular epithelial marker antibodies include CK8 / 18, CK (pan).

[0046] During the research and development process, the applicant used CK8 / 18+CK5 and CK(pan)+CK5 to perform immunohistochemical double staining on the same breast cancer tissue chip. Figure 3 The results of breast cancer tissue microarray staining are shown. The left image shows a mixed primary antibody of CK8 / 18 and CK5, while the right image shows a mixed primary antibody of CKpan and CK5. Figure 3 The results showed no significant difference between the two antibody combinations in detecting 90 breast cancers. The applicant discovered that CK(pan) also stained the myoepithelium in several breast cancer tissues, causing staining overlap and interference, leading to blurred myoepithelial staining boundaries and hindering the distinction between carcinoma in situ and invasive carcinoma.

[0047] Therefore, the preferred breast cancer tumor cell marker is CK8 / 18.

[0048] In some embodiments, in order to avoid interference from the antibodies themselves during the staining process, the antibodies in the mixed primary antibody are all monoclonal antibodies.

[0049] Since glandular epithelial markers and Ki-67 are co-expressed in invasive breast cancers that require counting, it is advantageous to identify and count cells expressing two different colors. In some embodiments, glandular epithelial markers and Ki-67 are detected using antibodies from different species.

[0050] In some embodiments, the muscle epithelial cell marker and the gland epithelial marker use antibodies of the same species. In this way, the muscle epithelial cell marker and the gland epithelial marker have the same color development, while the ki-67 has a different color development. For example, the ki-67 uses a mouse monoclonal antibody, while the muscle epithelial cell marker and the gland epithelial marker use a rabbit monoclonal antibody, or the ki-67 uses a rabbit monoclonal antibody, while the muscle epithelial cell marker and the gland epithelial marker use a mouse monoclonal antibody.

[0051] In some embodiments, the muscle epithelial cell marker and the ki-67 use antibodies of the same species, so that the muscle epithelial cell marker and the ki-67 have the same color development, while the gland epithelial marker has a different color development. For example, the ki-67 and the muscle epithelial cell marker use a mouse monoclonal antibody, while the gland epithelial marker uses a rabbit monoclonal antibody, or the ki-67 and the muscle epithelial cell marker use a rabbit monoclonal antibody, while the gland epithelial marker uses a mouse monoclonal antibody.

[0052] In some embodiments, the ki-67 uses a rabbit monoclonal antibody, the gland epithelial marker antibody CK8 / 18 uses a mouse monoclonal antibody, and the muscle epithelial marker antibody CK5 uses a rabbit monoclonal antibody.

[0053] Since both ki-67 and CK5 use rabbit monoclonal antibodies, their staining colors are consistent. However, ki-67 is located in the nucleus of infiltrating cancer cells, while CK5 is located in the cytoplasm of in situ cancer and normal breast epithelial cells, and is not expressed in infiltrating cancer cells. Therefore, it generally does not interfere with the counting of ki-67 slides.

[0054] In the prior art, due to the mutual influence and interference of the antibodies (including primary antibodies and secondary antibodies) themselves, the accuracy of ki-67 localization and the positive counting results are easily affected. Therefore, the current detection of ki-67 in breast infiltrating carcinoma generally uses single staining, and less mixed antibodies with the rest of the primary antibodies.

[0055] However, after the applicant studied the specificity of the antibodies and the detection system, it was found that the combination of ki-67 (clone MXR002), CK8 / 18 (mouse monoclonal antibody (MX004+MX035)), and CK5 (rabbit monoclonal antibody (EP24)) with mixed secondary antibodies (goat anti-mouse and goat anti-rabbit) has strong specificity, accurate localization, and basically no mutual interference between antibodies. Finally, the ki-67 color development result is similar to single staining. At the same time, the above double staining and three markers can significantly save the staining time and improve the accuracy of ki-67 counting.

[0056] The applicant has screened and studied the secondary antibodies and color development systems matched with the primary antibodies.

[0057] In some embodiments, the kit comprises a chromogenic solution, and the chromogenic solution comprises an AP-Red chromogenic solution and a DAB chromogenic solution.

[0058] In some embodiments, Ki-67 and CK5 use rabbit monoclonal antibodies, and are colored with an HRP-DAB system (brown); and CK8 / 18 uses mouse monoclonal antibodies, and is colored with an AP-RED system (red).

[0059] CK8 / 18, CK5 and Ki-67 are colored using an HRP detection system and an AP-RED detection system, and the results are shown in FIG. 2. The left panel is CK5-HRP (brown), Ki-67-HRP (brown), and CK8 / 18-AP (red); and the right panel is CK8 / 18-HRP (brown), CK5-AP (red), and Ki-67-AP (red). The left panel is more clear and can more obviously distinguish tumor cells and Ki-67-positive tumor cells; and the counting of Ki-67 is more convenient. Figure 4

[0060] In some embodiments, Ki-67 and CK5 are colored using a DAB chromogenic solution, and CK8 / 18 is colored using an AP-RED chromogenic solution, and the specific results are shown in FIG. 3. Figure 5 FIG. 3 is a comparison chart of different color development sequences of DAB and AP-RED.

[0061] In the color development solution research, it is found that the order of adding the color development solution has an effect on the results of Ki-67 positive color development. First, DAB color development solution is added, and then AP-RED color development solution is added; in this way, Ki-67 is colored first, and the number of Ki-67 positive counts is obviously higher than that when AP-RED color development solution is added first, and the staining of CK8 / 18 and CK5 does not differ much. When AP-RED color development solution is added first, and then DAB color development solution is added, the number of Ki-67 positive color development is affected, and missed detection is caused.

[0062] In some embodiments, the kit further comprises an antigen repair solution. Breast cancer tissue is paraffin-embedded after formalin repair, and the protein configuration is affected, and needs to be recovered by antigen modification. The main methods of antigen repair include citric acid hot repair, EDTA hot repair and microwave repair.

[0063] In some embodiments, EDTA hot repair is used.

[0064] In some embodiments, the kit further comprises an endogenous peroxidase blocking agent H2O2. In order to avoid the reaction of endogenous peroxidase on the tissue with DAB, an endogenous peroxidase blocking agent is used for blocking before using the primary antibody.

[0065] ​In some embodiments, the kit further comprises hematoxylin staining solution. Hematoxylin can develop blue background color for cell nucleus, and after the development of the color solution is completed, hematoxylin is added again, so that the unstained tissue cell nucleus has a light blue layer, which is convenient for identification.

[0066] Embodiments

[0067] In order to further clearly illustrate and describe the technical solutions of the present application, the following non-limiting embodiments are provided for reference.

[0068] In this embodiment, the mixed first antibody Ki-67 uses rabbit monoclonal antibody with clone MXR002, the adenocarcinoma marker antibody uses CK8 / 18 mouse monoclonal antibody with clone MX004+MX035, and the myoepithelial marker antibody uses CK5 rabbit monoclonal antibody with clone EP24. The mixed second antibody includes HPR-goat anti-rabbit secondary antibody and AP enzyme-labeled secondary antibody, and the color developing solution includes DAB color developing solution and AP-RED color developing solution.

[0069] In this embodiment, the breast tissue section is prepared by the following method: after the breast cancer tissue block is cut into sections, the thickness is set to 3 μm, the continuous sections are floated in cold water and naturally expanded, and then the separated sections are transferred to warm water at 45°C for 30 seconds. The sections are attached to the slides treated with polylysine in order, and the prepared tissue chip is placed in a 65°C oven for 2 hours, cooled to room temperature, and stored in a -4°C refrigerator.

[0070] In this embodiment, the DAB color developing solution needs to be prepared temporarily before use. When a small amount of precipitate appears during preparation, it can be filtered and used, which does not affect the staining effect and quality. The preparation method is as follows: mix the buffer and chromogen in a ratio of 20:1. The prepared DAB color developing solution is stored in the dark at 2-8°C and is effective within 7 days.

[0071] In this embodiment, the AP-RED color developing solution is prepared temporarily before use, and the buffer and chromogen are mixed in a ratio of 1:1. The prepared color developing solution is stored in the dark and is effective within 30 minutes.

[0072] In this embodiment, the antigen repair method uses EDTA heat repair method.

[0073] In this embodiment, room temperature refers to 25°C.

[0074] In this embodiment, the interpretation rules for breast tissue types are as follows:

[0075]

[0076] After the determination of the breast tissue type is completed, all tumor cells in the invasive breast cancer region (count the cells with red cytoplasm and blue or brown nuclei) and Ki-67 positive tumor cells (count the cells with red cytoplasm and brown nuclei) are counted.

[0077] Example 1 Immunohistochemical staining of breast cancer

[0078] Take one breast cancer tissue section, deparaffinize with xylene for 3 times, 6 minutes each time, hydrate in 100%, 100%, 95%, 85% gradient ethanol for 3 minutes each time, and finally rinse with tap water. Perform antigen retrieval, then place the section in a wet box, rinse with PBS for 3 x 3 minutes. Add 3% H2O2 and incubate for 10 minutes, rinse with PBS for 3 x 3 minutes.

[0079] After spin-drying treatment of the breast cancer tissue staining section, add mixed primary antibody and incubate at room temperature for 1 hour, rinse with PBS for 3 x 3 minutes, add mixed secondary antibody and incubate at room temperature for 15-30 minutes, rinse with PBS for 3 x 3 minutes, spin off the PBS, develop with DAB developing solution, rinse with PBS for 2 x 3 minutes; spin off the PBS, add freshly prepared AP-Red developing solution for development, rinse with PBS for 2 x 3 minutes; stain with hematoxylin for 25 seconds, return to blue with PBS for 30 seconds, dehydrate in 85%-95%-100%-100% alcohol gradient for 3 minutes each time, finally clear with xylene for 3 minutes, and mount with neutral balsam.

[0080] The staining results are shown in Figure 6 The breast cancer tissue section includes both breast carcinoma in situ (with a relatively complete brown gland periphery) and breast invasive carcinoma tissue (without a brown gland periphery, but part of the cells in the gland have cytoplasm and nuclei staining).

[0081] The experimenter combines the morphological characteristics of the breast tissue, first excludes the carcinoma in situ region in the section according to the staining results of CK8 / 18, and confirms the region of breast invasive carcinoma; then further counts the Ki-67 positive tumor cells in the invasive breast cancer region (cells with red cytoplasm and brown nuclei). Cells with only brown nuclei are not counted. Figure 6 In the experiment, it can be simply and clearly determined that in the breast invasive carcinoma region, Ki-67 > 30%, which is high expression.

[0082] Example 2 Immunohistochemical staining of breast

[0083] Take 1 slice of breast lobular hyperplasia tissue, conventional xylene dewaxing 3 times, 6 minutes each time, 100%, 100%, 95%, 85% gradient ethanol in hydration, 3 minutes each time, finally tap water rinse. Antigen repair is performed, then the slice is placed in a wet box, PBS is rinsed 3x3 minutes. Add 3% H2O2 and incubate for 10 minutes, PBS is rinsed 3x3 minutes.

[0084] After spin-drying treatment of the breast tissue stained slice, add mixed primary antibody, incubate at room temperature for 1 hour, PBS is rinsed 3x3 minutes, add mixed secondary antibody and incubate at room temperature for 15-30 minutes, PBS is rinsed 3x3 minutes, spin off PBS, develop color with DAB developing solution, PBS is rinsed 2x3 minutes; spin off PBS, add freshly prepared AP-Red developing solution to develop color, PBS is rinsed 2x3 minutes; hematoxylin counterstaining for 25 seconds, PBS is returned to blue for 30 seconds, sequentially dehydrated according to the alcohol gradient of 85%-95%-100%-100% for 3 minutes, finally xylene transparent for 3 minutes, neutral resin mounting.

[0085] The staining results are: CK5 and CK8 / 18 respectively stain the cytoplasm of the breast peripheral myoepithelium and the gland, the CK5 myoepithelial cells of the gland periphery show complete brown color, and the cytoplasm of the gland epithelial cells shows brown color. The nucleus is blue stained with hematoxylin, and the nucleus of Ki-67 is brown.

[0086] Example 3 Immunohistochemical staining of breast carcinoma in situ

[0087] Take 1 slice of breast carcinoma in situ tissue, conventional xylene dewaxing 3 times, 6 minutes each time, 100%, 100%, 95%, 85% gradient ethanol in hydration, 3 minutes each time, finally tap water rinse. Antigen repair is performed, then the slice is placed in a wet box, PBS is rinsed 3x3 minutes. Add 3% H2O2 and incubate for 10 minutes, PBS is rinsed 3x3 minutes.

[0088] After spin-drying treatment of the breast tissue stained slice, add mixed primary antibody, incubate at room temperature for 1 hour, PBS is rinsed 3x3 minutes, add mixed secondary antibody and incubate at room temperature for 15-30 minutes, PBS is rinsed 3x3 minutes, spin off PBS, develop color with DAB developing solution, PBS is rinsed 2x3 minutes; spin off PBS, add freshly prepared AP-Red developing solution to develop color, PBS is rinsed 2x3 minutes; hematoxylin counterstaining for 25 seconds, PBS is returned to blue for 30 seconds, sequentially dehydrated according to the alcohol gradient of 85%-95%-100%-100% for 3 minutes, finally xylene transparent for 3 minutes, neutral resin mounting.

[0089] The above staining results show that: the primary antibody uses Ki-67 (clone MXR002), CK8 / 18 (MX004+MX035), CK5 (EP24) combination, and cooperates with mixed secondary antibody (goat anti-mouse and goat anti-rabbit), the specificity is strong, the positioning is accurate, and the antibodies basically do not interfere with each other, and finally the Ki-67 color development result is similar to that of single staining. The three markers (Ki-67, CK5 and CK8 / 18) all show expression in breast carcinoma in situ. CK5 and CK8 / 18 stain the peripheral myoepithelium of breast carcinoma in situ and the cytoplasm in the gland. The myoepithelial cells of the gland periphery are complete brown, and the cytoplasm of the epithelial cells in the gland is brown, and the nucleus is the nucleus of Ki-67.

[0090] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of the present application, the patent protection scope of the present application should not be limited. Any technical solution obtained by replacing or modifying the equivalent structure or equivalent process based on the essential concept of the present application, using the content described in the specification and drawings of the present application, and directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, etc. are all included in the patent protection scope of the present application.

Claims

1. An immunohistochemical double staining triple marker kit for breast invasive carcinoma Ki-67 index interpretation, characterized in that, The kit comprises: mixed primary antibodies; the mixed primary antibodies comprise mouse antibodies and rabbit antibodies; the mixed primary antibodies comprise muscle epithelial marker antibodies, gland epithelial marker antibodies and Ki-67 antibodies; the muscle epithelial marker antibodies and the gland epithelial marker antibodies are located in the cytoplasm and / or cell membrane; Ki-67 adopts a rabbit monoclonal antibody, the gland epithelial marker antibody adopts a CK8 / 18 mouse monoclonal antibody, and the muscle epithelial marker antibody adopts a CK5 rabbit monoclonal antibody; the kit further comprises mixed secondary antibodies and a color developing solution; the mixed secondary antibodies comprise HPR-goat anti-rabbit secondary antibodies and AP enzyme-labeled secondary antibodies, and the color developing solution comprises DAB color developing solution and AP-RED color developing solution.

2. The kit of claim 1, wherein In the color developing step, the DAB color developing solution is used to develop Ki-67 and CK5, and the AP-RED color developing solution is used to develop CK8 / 18.

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