Multi-immunohistochemical overdyeing kit for identifying breast micro-invasive cancer and dyeing method

Abstract

The invention relates to the technical field of biological medicine, in particular to a multiple immunohistochemical overdyeing kit for identifying breast micro-invasive cancer and a dyeing method. The multi-immunohistochemical overdyeing kit for identifying the breast micro-invasive cancer comprises a mixed primary antibody, a mixed secondary antibody, a first immunohistochemical reagent, a second immunohistochemical reagent, a third immunohistochemical reagent, a fourth immunohistochemical reagent, a fourth immunohistochemical reagent and a fifth immunohistochemical reagent, the independent primary antibody is an SMA antibody; the mixed second antibody comprises an IgG antibody which is matched with a p63 antibody and marked by HRP and an IgG antibody which is matched with a CK5 / 6 antibody and marked by AP; the independent second antibody is an IgG (Immunoglobulin G) antibody which is matched with the SMA antibody and is marked by HRP; the color developing agent comprises a DAB color developing solution, a Fast Red color developing solution and a TMB color developing solution; an antigen repair liquid; a hematoxylin restaining solution; and returning blue liquid. According to the kit and the staining method, three markers on breast micro-invasive cancer tissues can be stained, and the infiltration state of the breast cancer, whether cancer cells break through a substrate or not and whether substrate cells are complete or not can be accurately represented.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a multiple immunohistochemical staining kit and a staining method for identifying microinvasive breast cancer. Background Art

[0002] Microinvasive breast cancer is defined as invasive breast cancer with a maximum diameter of 1 mm or less, which can be single or multifocal. The morphological characteristics are small tumor cell clusters or single cell infiltration. It is most common in ductal carcinoma in situ (DCIS), specifically DCIS with microinvasion. Accurate diagnosis of microinvasive breast cancer from DCIS is crucial for optimizing treatment and improving clinical prognosis.

[0003] Immunohistochemistry is a method used to detect specific antigens in tissue sections. By using specific antibodies to bind to target antigens, protein expression in cells or tissues can be visualized under a microscope. Multiplex Immunohistochemistry (mIHC) can detect multiple antigens on the same tissue section at the same time, which helps to understand the interactions and expression patterns between different proteins. By marking different colors and characterizing the spatial information between proteins, rare tissue samples can be saved, while conventional IHC can usually only detect one marker. Because the invasive lesions of microinvasive breast cancer are very small, it is difficult for pathologists to obtain accurate judgments from a single myoepithelial immune marker, which can easily lead to misjudgments or deviations in the accuracy of the judgment. Multiple immunohistochemistry is expected to improve the accuracy of judgments. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a multiple immunohistochemical staining kit and staining method for identifying microinvasive breast carcinoma, aiming to solve the technical problem that microinvasive breast carcinoma is difficult to judge in the prior art.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first aspect of the present invention provides a multiple immunohistochemical staining kit for identifying microinvasive breast cancer, comprising:

[0007] Mixed primary antibodies, including: p63 antibody and CK5 / 6 antibody;

[0008] Independent primary antibody: SMA antibody;

[0009] Mixed secondary antibodies, including: HRP-labeled IgG antibody compatible with p63 antibody, AP-labeled IgG antibody compatible with CK5 / 6 antibody;

[0010] Independent secondary antibody: HRP-labeled IgG antibody compatible with SMA antibody;

[0011] Color developing agents, including: DAB color developing solution, Fast Red color developing solution and TMB color developing solution;

[0012] Antigen retrieval solution; Hematoxylin counterstaining solution; Blueing solution.

[0013] In the multiple immunohistochemical staining kit for identifying microinvasive breast cancer, the antigen repair solution is EDTA antigen repair solution.

[0014] A second aspect of the present invention provides a method for staining microinvasive breast cancer multiple immunohistochemistry, which uses the multiple immunohistochemistry staining kit for identifying microinvasive breast cancer as described above, and comprises the following steps:

[0015] S001. Dewax the paraffin tissue sections and soak them in gradient alcohol until hydrated;

[0016] S002. Antigen retrieval followed by peroxidase blocking;

[0017] S003. Incubate with mixed primary antibodies, and then incubate with mixed secondary antibodies;

[0018] S004. DAB and Fast Red color developing solutions were used for staining respectively;

[0019] S005. Antigen retrieval again;

[0020] S006. Incubate with an independent primary antibody, then incubate with an independent secondary antibody;

[0021] S007. Staining with TMB colorimetric solution;

[0022] S008. Use hematoxylin counterstaining solution to counterstain the cell nucleus, use bluing solution to re-blue, dehydrate, dry, and seal the slides.

[0023] The multiple immunohistochemical staining method for microinvasive breast cancer, wherein the antigen repair of S002 and S005, comprises the following steps: using EDTA repair solution to perform antigen repair, placing the tissue section in the repair solution in a slightly boiling state, and repairing for 10 minutes; after the repair is completed, cooling to room temperature, fully rinsing with distilled water, and then rinsing with PBS 3 times, each time for 5 minutes.

[0024] The multiple immunohistochemical staining method for microinvasive breast cancer, wherein the S002 blocks peroxidase, comprises the following steps: placing the tissue section in a 3% H2O2 solution, incubating at room temperature for 10 minutes, and then washing with PBS 3 times, each time for 5 minutes.

[0025] The multiple immunohistochemical staining method for microinvasive breast carcinoma, wherein, in S003, the incubation condition for the mixed primary antibody is incubation at room temperature for 1 hour, and the incubation condition for the mixed secondary antibody is incubation at room temperature for 15 minutes.

[0026] The multiple immunohistochemical staining method for microinvasive breast cancer, wherein, in S004, the staining steps of DAB color developing solution are as follows: dripping DAB color developing substrate on the tissue section, and then dripping DAB color developing solution, controlling the reaction time under a microscope, the color developing time is 2 to 5 minutes, until the brown expression is obvious, and then rinsing in water to stop the color development.

[0027] The multiple immunohistochemical staining method for microinvasive breast cancer, wherein, in S004, the staining step of Fast Red color developing solution is as follows: DAB color developing substrate is added dropwise on the tissue section, and then Fast Red color developing solution is added dropwise, and the reaction time is controlled under a microscope, and the color development time is 4.5 to 5 minutes, until the red expression is obvious, and then the section is placed in water and fully rinsed to stop the color development.

[0028] The multiple immunohistochemical staining method for microinvasive breast carcinoma, wherein, in S003, the incubation condition of the independent primary antibody is incubation at room temperature for 1 hour, and the incubation condition of the independent secondary antibody is incubation at room temperature for 15 minutes.

[0029] The multiple immunohistochemical staining method for microinvasive breast cancer, wherein, in S007, the staining step of the TMB color developing solution is as follows: TMB color developing solution is dripped onto the tissue section, the reaction time is controlled under a microscope, the color developing time is 4.5 to 5 minutes, until the green expression is obvious, and then the section is fully rinsed in water to stop the color development.

[0030] Beneficial effects: The kit and the corresponding staining method of the present invention can achieve staining of three epithelial marker antibodies (p63, SMA and CK5 / 6) on the same tissue section through multiple immunohistochemistry, and present different colors. By observing the staining results of myoepithelial and glandular epithelial cells, accurate positioning can be achieved, thereby effectively judging whether tumor cells have broken through the basal tissue and the infiltration level of breast cancer, and improving the accuracy of judging microinvasive breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram showing the localization pattern of CK5 / 6, p63 and SMA in mammary ductal epithelial cells.

[0032] Figure 2 The following are photos of breast hyperplasia tissue sections of Comparative Examples 3-6 under a 100x microscope.

[0033] Figure 3The following are photos of the breast microinvasive carcinoma tissue sections of Comparative Examples 3-6 under a 100x microscope.

[0034] Figure 4 The following are photos of the invasive breast cancer tissue sections of Comparative Examples 3-6 under a 100x microscope.

[0035] Figure 5 The following are photos of breast hyperplasia tissue sections of Example 1, Comparative Example 1 and Comparative Example 2 under a 100x microscope.

[0036] Figure 6 The following are photos of breast microinvasive carcinoma tissue sections of Example 1, Comparative Example 1 and Comparative Example 2 under a 100x microscope.

[0037] Figure 7 The following are photos of invasive breast cancer tissue sections of Example 1, Comparative Example 1 and Comparative Example 2 under a 100x microscope. DETAILED DESCRIPTION

[0038] The present invention provides a multiple immunohistochemical staining kit and staining method for identifying microinvasive breast cancer. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] The present invention provides a multiple immunohistochemical staining kit for identifying microinvasive breast cancer, comprising:

[0040] Mixed primary antibodies, including: p63 antibody and CK5 / 6 antibody;

[0041] Independent primary antibody: SMA antibody;

[0042] Mixed secondary antibodies, including: HRP-labeled IgG antibody compatible with p63 antibody, AP-labeled IgG antibody compatible with CK5 / 6 antibody;

[0043] Independent secondary antibody: HRP-labeled IgG antibody compatible with SMA antibody;

[0044] Color developing agents, including: DAB color developing solution, Fast Red color developing solution and TMB color developing solution;

[0045] Antigen retrieval solution; Hematoxylin counterstaining solution; Blueing solution.

[0046] Specifically, the type of secondary antibody can be selected according to the animal species of the primary antibody.

[0047] When this kit is used, the p63 antibody and its secondary antibody, combined with the DAB color developing solution, locate the cell nucleus and form a brown color;

[0048] CK5 / 6 antibody and its secondary antibody, combined with Fast Red colorimetric solution, locate the basal duct epithelial cells and display red;

[0049] SMA antibody and its secondary antibody, combined with TMB colorimetric reagent, locate the myoepithelial cells and display green.

[0050] This kit has developed a triple-staining immunohistochemical staining kit for three different myoepithelial markers in the traditional single immunohistochemical staining and cocktail double immunohistochemical staining methods. In DICS and invasive breast cancer, the integrity of single myoepithelial expression cannot fully distinguish whether there is invasive breast cancer in DICS, especially in breast cancer with microinfiltration of tissues, which is often ignored by pathologists because the tissue infiltration foci are too small, affecting the subsequent clinical medication and treatment. The present invention uses three typical myoepithelial markers (CK5 / 6, p63, SMA) to achieve marking three different myoepithelial cells in the same slice, which can simultaneously characterize the infiltration state of breast cancer, and can better judge whether there are cancer cells breaking through the basement and whether the basement cells are intact. Multiple labeling helps to better understand the role of myoepithelial cells in the tumor microenvironment. The multiple staining images provided by mIHC can clearly show the relationship between myoepithelial cells and other cells, and enhance the understanding of tissue structure and pathological characteristics. Secondly, by marking myoepithelial cells, the boundary between the tumor and the surrounding tissue can be observed more clearly, and the infiltration and invasiveness of the tumor can be evaluated. This is of great significance for judging the grade and prognosis of the tumor. By analyzing the expression of myoepithelial cells, mIHC can help distinguish different types of breast cancer (such as invasive ductal carcinoma and ductal carcinoma in situ) and improve the accuracy of diagnosis. At the same time, the status of myoepithelial cells (such as absence or abnormal proliferation) is closely related to the prognosis of breast cancer. mIHC can help evaluate these markers and provide more accurate information for patients' prognosis.

[0051] This kit can make pathological diagnosis more precise and accurate, reduce the difficulty of reading films, and can distinguish the degree of infiltration of tissues according to different colors in the same field of view, reducing the number of sections. It has good application prospects and feasibility in clinical diagnosis.

[0052] Example 1

[0053] A multiple immunohistochemical staining method for microinvasive breast cancer comprises the following steps:

[0054] A001. Prepare a paraffin section of invasive breast cancer and a paraffin section of microinvasive breast cancer respectively; dewax the paraffin tissue sections with xylene for 3 times, 5 minutes each time; perform gradient alcohol treatment in descending order of ethanol concentration, where the ethanol concentration is 100%, 100%, 95%, 85%, and hydrate for 3 minutes at each concentration; then rinse thoroughly with tap water until there is no obvious trace of xylene residue on the tissue surface;

[0055] A002. Perform antigen repair using EDTA antigen repair solution (pH=7.0): Heat the solution in an induction cooker water bath until the solution is completely boiling. Place the slide in the pot and adjust the voltage of the induction cooker to keep the repair solution at a slight boiling state for 20 minutes. After the repair is completed, cool to room temperature and rinse thoroughly with distilled water. Rinse with PBS 3 times for 5 minutes each time.

[0056] A003. Block endogenous peroxidase; incubate tissue sections in 3% H2O2 at room temperature for 10 minutes; then rinse with PBS 3 times, 5 minutes each time;

[0057] A004. Multiple immunohistochemical staining process, using mixed primary antibodies, dripping on the same section for staining, the mixed primary antibodies include rabbit polyclonal antibody against p63 and mouse monoclonal antibody against CK5 / 6, incubating at room temperature for 1 hour, and washing thoroughly with PBST 3 times, 15 minutes each time;

[0058] A005. Add mixed secondary antibodies, including goat anti-rabbit IgG labeled with HRP horseradish peroxidase and goat anti-mouse IgG labeled with AP alkaline phosphatase, incubate at room temperature for 15 minutes, and then rinse with PBST three times, 15 minutes each time;

[0059] A006. Add DAB color developing solution to the tissue section and control the reaction time under a microscope. The color development time is 2 to 5 minutes until the brown color is obvious. Then rinse thoroughly in water to stop the color development.

[0060] A007. Add Fast Red color developing solution to the tissue section and control the reaction time under a microscope. The color development time is about 5 minutes until the red color is clearly expressed. Then rinse thoroughly in water to stop the color development.

[0061] A008. Perform antigen retrieval again using EDTA antigen retrieval solution, and then block endogenous peroxidase again;

[0062] A009. Add independent primary antibody, rabbit polyclonal antibody against SMA, to the tissue sections, incubate at room temperature for 1 hour, and then rinse with PBST three times, 15 minutes each time;

[0063] A010. Add independent secondary antibody, HRP-labeled goat anti-rabbit IgG antibody, incubate at room temperature for 15 minutes, and then rinse with PBST three times, 15 minutes each time;

[0064] A011. Add TMB colorimetric solution to the tissue section and control the reaction time under a microscope. The color development time is about 5 minutes until the red color is clearly expressed. Then rinse thoroughly in water to stop the color development.

[0065] A012. Use hematoxylin counterstaining solution to counterstain the cell nucleus, then use bluing solution for 1 minute, and use 85% alcohol, 95% alcohol, anhydrous ethanol 1, and anhydrous ethanol 2 for dehydration in sequence. After the slices are dry, add sealing medium to seal the slices.

[0066] Comparative Example 1

[0067] A multiple immunohistochemistry method for microinvasive breast cancer is different from Example 1 in that step A008 is not performed, and EDTA antigen repair solution is not used again for antigen repair, and then endogenous peroxidase is not blocked again.

[0068] Comparative Example 2

[0069] A multiple immunohistochemical staining method for microinvasive breast cancer is different from Example 1 in that a rabbit polyclonal antibody against calponin is used instead of a rabbit polyclonal antibody against p63.

[0070] Comparative Example 3

[0071] A method for staining microinvasive breast cancer comprises the following steps: preparing a paraffin section of invasive breast cancer, a microinvasive ductal carcinoma in situ and a breast hyperplasia, using a Sakura fully automatic HE staining instrument (Tissue-Tek Plus) for staining.

[0072] Comparative Example 4

[0073] A method for staining microinvasive breast cancer comprises the following steps:

[0074] A001. Prepare a paraffin section of invasive breast cancer, a microinvasive breast cancer, and a breast hyperplasia, dewax them, and soak them in gradient alcohol until they are hydrated;

[0075] A002. Use EDTA repair solution to perform antigen repair, and then block peroxidase;

[0076] A003. Add primary antibody, mouse monoclonal antibody against CK5 / 6, incubate at room temperature for 1 hour, and rinse thoroughly with PBST 3 times, 15 minutes each time;

[0077] A004. Add secondary antibody, HRP-labeled goat anti-mouse and goat anti-rabbit mixed IgG antibody, incubate at room temperature for 30 minutes, and then rinse with PBST three times, 15 minutes each time;

[0078] A005. Use DAB colorimetric solution for staining;

[0079] A006. Use hematoxylin counterstaining solution to counterstain the cell nucleus, use bluing solution to reblue, dehydrate, dry and seal the slides.

[0080] Comparative Example 5

[0081] A method for staining microinvasive breast cancer, which differs from Comparative Example 4 in that a primary antibody is a rabbit polyclonal antibody against p63.

[0082] Comparative Example 6

[0083] A method for staining microinvasive breast cancer, which differs from Comparative Example 4 in that a primary antibody is a rabbit polyclonal antibody against SMA.

[0084] The tissue sections prepared in the above embodiments and comparative examples were scanned using a section scanning instrument, and the results are shown in the accompanying drawings.

[0085] Figure 1 This is a diagram showing the localization pattern of CK5 / 6, p63 and SMA in mammary ductal epithelial cells.

[0086] Figure 2 Part (A) is the breast hyperplasia tissue section of Comparative Example 3; Part (B) is the breast hyperplasia tissue section of Comparative Example 4; Part (C) is the breast hyperplasia tissue section of Comparative Example 5; and Part (D) is the breast hyperplasia tissue section of Comparative Example 6.

[0087] Figure 3 Part (A) is a breast microinvasive carcinoma tissue section of Comparative Example 3; Part (B) is a breast microinvasive carcinoma tissue section of Comparative Example 4; Part (C) is a breast microinvasive carcinoma tissue section of Comparative Example 5; and Part (D) is a breast microinvasive carcinoma tissue section of Comparative Example 6.

[0088] Figure 4 Part (A) is the invasive breast cancer tissue section of Comparative Example 3; Part (B) is the invasive breast cancer tissue section of Comparative Example 4; Part (C) is the invasive breast cancer tissue section of Comparative Example 5; and Part (D) is the invasive breast cancer tissue section of Comparative Example 6.

[0089] from Figure 2-Figure 4 It can be seen that no matter whether a fully automatic HE staining instrument is used or CK5 / 6, p63 and SMA are individually labeled and colored, the staining results of breast hyperplasia tissue sections, breast microinvasive carcinoma tissue sections and breast microinvasive carcinoma tissue sections obtained have certain defects.

[0090] In particular, the results of staining of breast microinvasive carcinoma tissue sections ( Figure 3 ) and the staining results of breast microinvasive carcinoma tissue sections ( Figure 4), the staining results obtained by various single immunohistochemical staining methods showed that the expression of p63 and CK5 / 6 had not completely disappeared in microinvasive breast cancer, and it was impossible to identify the integrity of the basement by single p63 and CK5 / 6 staining, especially in the microinvasive area of ​​the breast, where the invasive lesions are relatively small, the single staining results are unclear, the staining effect is poor, and it is easy to lead to missed diagnosis.

[0091] Figure 5 Part (A) is the breast hyperplasia tissue section of Example 1; Part (B) is the breast hyperplasia tissue section of Comparative Example 1; and Part (C) is the breast hyperplasia tissue section of Comparative Example 2.

[0092] Figure 6 Part (A) is the breast microinvasive carcinoma tissue section of Example 1; Part (B) is the breast microinvasive carcinoma tissue section of Comparative Example 1; and Part (C) is the breast microinvasive carcinoma tissue section of Comparative Example 2.

[0093] Figure 7 Part (A) is the invasive breast cancer tissue section of Example 1; Part (B) is the invasive breast cancer tissue section of Comparative Example 1; and Part (C) is the invasive breast cancer tissue section of Comparative Example 2.

[0094] Figure 5-Figure 7 Part (A) in the figure is the tissue section prepared in Example 1, with CK5 / 6 showing red, p63 showing brown and SMA showing green, marking the expression of different epithelial markers in the mammary duct respectively. By developing the color of three different myoepithelial markers, the infiltration state of breast cancer, whether cancer cells have broken through the basal layer, and whether the basal cells are intact can be simultaneously characterized.

[0095] Specifically, Figure 5 In (A), we can clearly see that in breast hyperplasia tissue, CK5 / 6 is expressed in red and located in the cytoplasm, p63 is expressed in brown and located in the nucleus, and SMA is expressed in green and located in the cytoplasm. The three different color antibody staining can clearly outline the basement membrane position and identify the integrity of the breast basement.

[0096] Figure 6 In (A), we can clearly see that in the micro-invasive breast cancer tissue, the basement gradually disappears, but it has not completely disappeared. In the same slice, CK5 / 6, p63 and SMA are co-localized, and the expression of CK5 / 6 and p63 in the breast basement membrane gradually decreases, with only individual and partial expression. This indicates that there may be cancer cells that break through the basement and form micro-invasive foci.

[0097] Figure 7In (A), it can be clearly seen that the breast basement membrane has completely disappeared in the invasive breast cancer tissue. CK5 / 6, p63 and SMA are expressed individually or not at all. This indicates that the cancer cells have completely broken through the basement membrane and formed invasive breast cancer.

[0098] Compared with Example 1, the experimental steps of antigen repair were reduced in Comparative Example 1. Figure 5-Figure 7 As can be seen from part (B), the antibody expression and localization of Comparative Example 1 and Example 1 are obviously consistent, but in Comparative Example 1, CK5 / 6 and p63 antigens cannot bind, which increases some nonspecific coloring, easily leading to misjudgment of cancer cells breaking through the substrate and misjudgment of incomplete basal cells, affecting the diagnosis results.

[0099] Compared with Example 1, in Comparative Example 2, the p63 antibody was changed to the Calponin antibody. Figure 5-Figure 7 As can be seen in part (C), after labeling with Calpnin antibody, other epithelial markers cannot be displayed in the same tissue and are covered by Calponin epithelial staining.

[0100] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and inventive concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. A multiple immunohistochemical staining kit for identifying microinvasive breast cancer, characterized in that: include: Mixed primary antibodies, including: p63 antibody and CK5 / 6 antibody; Independent primary antibody: SMA antibody; Mixed secondary antibodies, including: HRP-labeled IgG antibody compatible with p63 antibody, AP-labeled IgG antibody compatible with CK5 / 6 antibody; Independent secondary antibody: HRP-labeled IgG antibody compatible with SMA antibody; Color developing agents, including: DAB color developing solution, Fast Red color developing solution and TMB color developing solution; Antigen retrieval solution; Hematoxylin counterstain; Blue liquid.

2. The multiple immunohistochemical staining kit for identifying microinvasive breast cancer according to claim 1, characterized in that: The antigen repair solution is EDTA antigen repair solution.

3. A method for multiple immunohistochemical staining of microinvasive breast cancer, characterized in that: The multiple immunohistochemical staining kit for identifying microinvasive breast cancer according to claim 1 or 2 comprises the following steps: S001. Dewax the paraffin tissue sections and soak them in gradient alcohol until hydrated; S002. Antigen retrieval followed by peroxidase blocking; S003. Incubate with mixed primary antibodies, and then incubate with mixed secondary antibodies; S004. DAB colorimetric solution and Fast Red colorimetric solution were used for staining respectively; S005. Antigen retrieval again, then blocking peroxidase; S006. Incubate with an independent primary antibody, then incubate with an independent secondary antibody; S007. Staining with TMB colorimetric solution; S008. Use hematoxylin counterstaining solution to counterstain the cell nucleus, use bluing solution to re-blue, dehydrate, dry, and seal the slides.

4. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: The antigen repair of S002 and S005 includes the following steps: using EDTA repair solution to perform antigen repair, placing the tissue sections in the repair solution in a slightly boiling state for 10 minutes; after the repair is completed, cooling to room temperature, fully rinsing with distilled water, and then rinsing with PBS for 3 times, each time for 5 minutes.

5. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: The peroxidase blocking method of S002 comprises the following steps: placing the tissue slice in a 3% H2O2 solution, incubating at room temperature for 10 minutes, and then washing with PBS for 3 times, each time for 5 minutes.

6. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: In the S003, the incubation condition of the mixed primary antibody is incubation at room temperature for 1 hour, and the incubation condition of the mixed secondary antibody is incubation at room temperature for 15 minutes.

7. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: In S004, the staining steps of DAB color developing solution are as follows: DAB color developing substrate is added dropwise to the tissue section, and then DAB color developing solution is added dropwise, and the reaction time is controlled under a microscope, and the color developing time is 2 to 5 minutes, until brown color is obviously expressed, and then the section is fully rinsed in water to stop color development.

8. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: In S004, the staining steps of Fast Red color developing solution are as follows: DAB color developing substrate is added to the tissue section, and then Fast Red color developing solution is added, and the reaction time is controlled under a microscope, and the color development time is 4.5 to 5 minutes, until the red expression is obvious, and then it is put into water for thorough washing to stop the color development.

9. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: In the S003, the incubation condition of the independent primary antibody is incubation at room temperature for 1 hour, and the incubation condition of the independent secondary antibody is incubation at room temperature for 15 minutes.

10. The method for multiple immunohistochemical staining of microinvasive breast cancer according to claim 3, characterized in that: In S007, the staining step of the TMB color developing solution is as follows: TMB color developing solution is dripped onto the tissue section, and the reaction time is controlled under a microscope, and the color developing time is 4.5 to 5 minutes, until the green expression is obvious, and then the section is fully rinsed in water to stop the color development.

Images