Cocktail sample immunohistochemical detection method

Abstract

The invention provides a cocktail sample immunohistochemical detection method, and belongs to the technical field of biological detection. According to the cocktail sample immunohistochemical detection method provided by the invention, two or more different antibodies are added on the same tissue slice or tissue chip, the characteristic that the antibodies are different in positioning is utilized, and a diaminobenzidine (DAB) developing solution is used, so that the display of a single staining detection result is realized; the problems that a detection method in the prior art is relatively high in slice and antibody consumption cost and relatively long in consumed time are solved. The cocktail sample immunohistochemical detection method provided by the invention has better detection accuracy, can be suitable for sample detection under various conditions, not only has high result consistency and strong sensitivity and specificity, but also reduces the experimental result error, reduces the usage amount, and saves the cost and time.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, in particular to a cocktail-like immunohistochemical detection method. Background Art

[0002] Tissue chip (TC), also known as tissue microarrays (TMAs), is a component of biochip technology, just like gene chip, protein chip and cell chip, and belongs to biological high-tech technology. Tissue chip is a new type of high-throughput, multi-sample research tool that arranges dozens or hundreds of tissue specimens of different individuals on a carrier according to a predetermined sequence for morphological or molecular biology experiments. Tissue chip technology has the advantages of large amount of information, small experimental error, strong comparability, time saving, labor saving, and large amount of cost saving. It can be combined with traditional pathological technology, histochemistry technology, immunohistochemistry technology (IHC), in situ hybridization technology (ISH), fluorescent nucleic acid in situ hybridization technology (FISH) and in situ PCR technology to perform morphological observation of normal or diseased tissue specimens, or to study different molecular levels such as specific genes and the proteins they express. Tissue chip technology has gradually been applied to scientific research, clinical practice, teaching, biological reagent testing, quality control and standardization in the fields of medicine, biology, zoology and botany.

[0003] Normally, anything that can be used as an antigen or hapten in a tissue or cell, such as proteins, peptides, amino acids, polysaccharides, phospholipids, receptors, enzymes, hormones, nucleic acids, and pathogens, can be detected with the corresponding specific antibodies. Each time an antigen is detected, a tissue slice or tissue chip (including paraffin and frozen slices) is required, and the antibody must be added once. Detecting antigens with different locations in the same tissue and cells is undoubtedly a waste of consumables and time. At the same time, there are certain differences in target tissues and cells in different slices; especially for some highly sensitive and specific antibodies, slight differences under the same conditions can better reflect different test results, such as changes in the cross-section of different slices or tissue chips, and differences in the number and thickness of target cells. The immunohistochemical results often show different intensities or quantities, and the accuracy of the detection still needs to be improved. Summary of the invention

[0004] The object of the present invention is to provide a cocktail-like immunohistochemical detection method, which has better detection accuracy and can be applied to sample detection under various conditions.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a cocktail-like immunohistochemical detection method, comprising the following steps:

[0007] S1. Dewaxing, antigen repairing and blocking the tissue sections to be examined in sequence;

[0008] S2, adding more than 2 antibodies;

[0009] S3, adding enzyme-labeled polymer secondary antibody, incubating and then washing;

[0010] S4, add DAB color developer, and wash with water after color development;

[0011] S5, add hematoxylin, wash with water until blue returns after differentiation, and complete immunohistochemical detection;

[0012] The tissue section to be examined is divided into a left half and a right half

[0013] A plurality of tissue points to be inspected are respectively arranged on the left half and the right half of the tissue section to be inspected;

[0014] In S2, the adding of two or more antibodies specifically comprises the following steps:

[0015] Add mixed antibody 1 to the left half of the tissue section to be tested, and add mixed antibody 2 to the right half of the tissue section to be tested, incubate and then wash;

[0016] The mixed antibody 1 and the mixed antibody 2 respectively contain more than two kinds of antibodies;

[0017] The types of antibodies in the mixed antibody 1 and the mixed antibody 2 are not completely the same.

[0018] Preferably, the cocktail immunohistochemistry detection method is a non-diagnostic method.

[0019] Preferably, the mixed antibody 1 or the mixed antibody 2 is obtained by vortex mixing the respective antibodies in equal proportions.

[0020] Preferably, the mixed antibody 1 contains Anti-ER and Anti-Her2;

[0021] The mixed antibody 2 contains Anti-PR and Anti-Her2.

[0022] Preferably, in S2, the adding of two or more antibodies specifically comprises the following steps:

[0023] Add antibody 1 to the left half of the tissue section to be tested, and add antibody 2 to the left half of the tissue section to be tested within 10 seconds and shake well;

[0024] Add antibody 1 to the right half of the tissue section to be tested, and add antibody 3 to the right half of the tissue section to be tested within 10 seconds and shake well, incubate and wash;

[0025] The antibody 1, antibody 2 and antibody 3 are antibodies against different target proteins.

[0026] Preferably, the antibody 1 is Anti-Her2;

[0027] The antibody 2 is Anti-PR;

[0028] The antibody 3 is Anti-ER.

[0029] The present invention also provides another cocktail-like immunohistochemical detection method, comprising the following steps:

[0030] S1. Dewaxing, antigen repairing and blocking the tissue sections to be examined in sequence;

[0031] S2, adding antibody 1 dropwise to the left and right halves of the tissue section to be tested, incubating and then washing;

[0032] S3, adding enzyme-labeled polymer secondary antibody, incubating and then washing;

[0033] S4, add DAB color developer, and wash with water after color development;

[0034] S5, adding antibody 2 to the left half of the tissue section to be tested, and adding antibody 3 to the right half of the tissue section to be tested, incubating and then washing;

[0035] S6, adding enzyme-labeled polymer secondary antibody, incubating and then washing;

[0036] S7. Add DAB colorimetric agent, wash with water after color development, and complete the immunohistochemical detection.

[0037] Preferably, the antibody 1 is Anti-Her2;

[0038] The antibody 2 is Anti-PR;

[0039] The antibody 3 is Anti-ER.

[0040] Preferably, the tissue slice to be tested is a microarray tissue chip or a common tissue slice containing a solid phase carrier;

[0041] The solid phase carrier is a glass slide;

[0042] Preferably, the slide is an anti-shedding immunohistochemistry slide, an immunofluorescence in situ hybridization slide or a poly-lysine slide.

[0043] Preferably, the dewaxing is performed by baking at a temperature of 50 to 70° C. for 20 to 40 minutes.

[0044] And / or, the reagent used for antigen retrieval is Tris-EDTA antigen retrieval buffer;

[0045] and / or, the pH of the Tris-EDTA antigen retrieval buffer is 8.0 to 9.0;

[0046] And / or, the sealing process comprises the following steps:

[0047] First, add H2O2-methanol to block endogenous peroxidase, incubate for 20-40 minutes, wash, add serum, and incubate for 5-15 minutes;

[0048] and / or, the concentration of H2O2-methanol blocking endogenous peroxidase is 2-4%;

[0049] And / or, the washing adopts Tween-20 PBS solution with a volume concentration of 0.05-0.2%.

[0050] Beneficial effects of the present invention:

[0051] The cocktail immunohistochemical detection method provided by the present invention achieves the display of single staining detection results by adding two or more different antibodies to the same tissue slice or tissue chip, utilizing their different positioning characteristics, and combining the use of diaminobenzidine (DAB) color developing solution. The detection method provided by the present invention has the characteristics of high result consistency, high sensitivity and specificity, while reducing the error of experimental results, reducing the amount of slices and antibodies used, and saving costs and time.

[0052] The cocktail-like immunohistochemical detection method of the present invention can be tested under different conditions and is applicable to cell membrane and cell nucleus immunohistochemistry (IHC), immunofluorescence (IF) detection, etc. of all target gene proteins localized in breast cancer, gastric cancer, colorectal cancer, endometrial cancer, ovarian cancer, etc. It can complete multiple antibody labeling at one time and is applicable to multiple fields such as tumor protein research or gene detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The pictures are the results of the cocktail immunohistochemistry test of the present invention, wherein: a: indirect method PR-B2 spot - tumor cell Her2 cell membrane +++ PR nucleus +++ 400X; b: front and back drop mixing method PR-B2 spot - tumor cell Her2 cell membrane +++ PR nucleus +++ 400X; c: direct shaking method PR-B2 spot - tumor cell Her2 cell membrane +++ PR nucleus +++ 400X;

[0054] Figure 2 This is the result of indirect detection of ER, PR and her-2 by immunohistochemistry of F048Br01 breast cancer tissue chip;

[0055] Figure 3 This is the result of direct shake and analysis of ER, PR, and her-2 in F048Br01 breast cancer tissue chip immunohistochemistry;

[0056] Figure 4 This is the result of the F048Br01 breast cancer tissue chip immunohistochemistry ER, PR, her-2 before and after the mixed drop method detection;

[0057] Figure 5 This is a schematic diagram of the F048Br01 breast cancer tissue chip structure, which contains 48 breast cancer spots, two spots in one case. DETAILED DESCRIPTION

[0058] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0059] Example 1 Cocktail Immunohistochemical Detection-Direct Shake Method

[0060] Antibodies Anti-Her2, Anti-ER, and Anti-PR were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Self-made breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0061] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0062] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0063] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0064] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0065] 5) Remove the serum, directly add 500ul of the same mass and proportion of Anti-ER and Anti-Her2 into the antibody sampling tube, use a vortex mixer to mix, hold the slide in the left hand, double-scoop the tissue microarray slide, add Anti-PR and Anti-Her2 to cover the left half of the tissue microarray chip, and add Anti-ER and Anti-Her2 to cover the right half, and put it in a 4°C refrigerator overnight;

[0066] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0067] 7) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 30 minutes, and prepare a negative control tissue chip under the same conditions;

[0068] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0069] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0070] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0071] 11) Conventional dehydration and transparency, neutral gum sealing.

[0072] 12) Observe the test slide: hold the label side in your left hand and read the slide from left to right and from top to bottom; the target cells are stained and located on the cell membrane. The results are specific, clear and contrasting (see the representative test results for the photos). Figure 1 c).

[0073] Example 2 Cocktail Immunohistochemistry Detection - Front and Back Mixing Method

[0074] Antibodies Anti-Her2, Anti-ER, and Anti-PR were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Self-made breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0075] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0076] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0077] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0078] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0079] 5) Remove serum, hold the slide with your left hand, scoop up the left half of the tissue microarray slide, first add Anti-Her2 to cover the microarray chip, then add Anti-PR within 10 seconds, shake the slide evenly; for the right half of the tissue microarray chip, first add Anti-Her2 to cover the microarray chip, then add Anti-ER within 10 seconds, shake the slide evenly, and put it in a 4°C refrigerator overnight;

[0080] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0081] 7) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 30 minutes, and prepare a negative control tissue chip under the same conditions;

[0082] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0083] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloration;

[0084] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0085] 11) Conventional dehydration and transparency, neutral gum sealing.

[0086] 12) Observe the test slide: hold the label side in your left hand and read the slide from left to right and from top to bottom; the target cells are stained and located on the cell membrane. The results are specific, clear and contrasting (see the representative test results for the photos). Figure 1 b).

[0087] Example 3 Cocktail Immunohistochemical Detection - Indirect Method

[0088] Antibodies Anti-Her2, Anti-ER, and Anti-PR were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Self-made breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0089] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0090] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0) at medium heat for 10 minutes until boiling, then turn off the heat for 5 minutes to keep warm and then turn to medium-low heat for 5 minutes to retrieval antigens, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0091] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0092] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0093] 5) Remove the serum, hold the slide with your left hand, scoop up the left and right tissue microarray chips on the tissue chip slide, and add the primary antibody Anti-Her2 at the same time, and place it in a 4°C refrigerator overnight;

[0094] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0095] 7) Add enzyme-labeled polymer secondary antibody and incubate in a 37°C incubator for 30 minutes. Prepare a negative control tissue chip under the same conditions.

[0096] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0097] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0098] 10) Hold the slide with your left hand, and add the primary antibodies Anti-PR and Anti-ER to the left and right tissue microarray chips on the tissue microarray slide at the same time; incubate at 37°C for 1 hour.

[0099] 11) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0100] 12) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 15 minutes, and prepare a negative control tissue chip under the same conditions;

[0101] 13) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0102] 14) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring (the representative test results are shown in the photo). Figure 1 a).

[0103] Experimental example

[0104] Instruments for immunohistochemistry experiments: constant temperature incubator, medical microwave oven, stainless steel pressure cooker, induction cooker, decolorization shaker, vortex mixer, histochemistry pen, pipette, antibody sampling tube.

[0105] Reagents used in immunohistochemistry experiments include anhydrous ethanol, xylene, EDTA repair solution, CB repair solution, PBS buffer, peroxidase blocker, primary antibody (ER, PR, Her2), Tween-20, TritonX-100; secondary antibody: ready-to-use HRP enzyme-labeled goat anti-mouse / rabbit IgG Enhanced Plus method, purchased from Suzhou Baidao Medical Technology Co., Ltd., product number: KIT-9901, histochemistry kit DAB color developer, hematoxylin, neutral gum.

[0106] Tissue microarray chips for immunohistochemistry experiments: homemade breast cancer and its matching adjacent tumor tissue combination chips, continuous sections, double slides (two continuous tissue wax slides are attached to each glass slide), 48 breast cancer tissue points on each chip, a total of 12 sections (including 6 test tissue chips and 6 negative control tissue chips), 288 breast cancer test tissue points, and 288 negative control test tissue points.

[0107] Experimental design: A homemade breast cancer and its matching adjacent tumor tissue combination chip was selected for continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), 48 breast cancer tissue points on each chip, slice thickness of 4 μm, a total of 12 chips were cut (including 6 test tissue chips and 6 negative control tissue chips), with 288 breast cancer test tissue points and 288 negative control test tissue points (276 actual effective tissue points).

[0108] First, 6 tissue chips were taken out, 3 of which were labeled with conventional monoclonal antibodies as positive control photos of the detection chip of the present invention, namely:

[0109] The first one uses estrogen receptor (ER) as the primary antibody for immunohistochemistry (the chip actually has 46 effective points);

[0110] In the second slide, progesterone receptor (PR) was selected as the primary antibody for immunohistochemistry (the chip actually had 46 effective points);

[0111] The third slide uses human epidermal growth factor receptor 2 (Her2) as the primary antibody for immunohistochemistry (the actual effective points on the chip are 46);

[0112] The remaining 3 images served as negative controls;

[0113] Then, the remaining 6 tissue chips were taken out, and 3 of them were subjected to immunohistochemical detection using the cocktail sample double-label single staining method:

[0114] The first tissue chip used the cocktail sample direct shaking method to perform primary antibody Her2 plus primary antibody ER and primary antibody Her2 plus primary antibody PR immunohistochemistry (the chip actually had 46 effective points);

[0115] The second tissue chip used the cocktail sample front-to-back mixing method to perform primary antibody Her2 plus primary antibody ER and primary antibody Her2 plus primary antibody PR immunohistochemistry (the chip actually had 46 effective points);

[0116] The third tissue chip used the cocktail indirect method to perform immunohistochemistry with primary antibody Her2 plus primary antibody ER and primary antibody Her-2 plus primary antibody PR (the chip actually had 46 valid points).

[0117] The remaining 3 images served as negative controls.

[0118] Experimental process:

[0119] 1 (conventional method), antibody Anti-ER was provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 658G3A2. Continuous sections of homemade breast cancer and its matching adjacent tumor tissue combination chip, double slides (two consecutive tissue wax slides were attached to each glass slide), 48 breast cancer tissue points on each chip.

[0120] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0121] Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0122] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0123] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0124] 5) Remove the serum, add the primary antibody Anti-ER (ready-to-use), and store in a refrigerator at 4°C overnight;

[0125] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0126] 7) Add enzyme-labeled polymer secondary antibody and incubate in a 37°C incubator for 30 minutes. Prepare a negative control tissue chip under the same conditions.

[0127] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0128] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0129] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0130] 11) Conventional dehydration and transparency, neutral gum sealing.

[0131] 12) Observe the test film: hold the label side in your left hand and read the film from left to right and from top to bottom; the target cells are colored and located in the cell nucleus. The results are specific, clear and contrasting.

[0132] 2 (conventional method), antibody Anti-PR was provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 278G8D6. Continuous sections of homemade breast cancer and its matching adjacent tumor tissue combination chip, double slides (two consecutive tissue wax slides were attached to each glass slide), 48 breast cancer tissue points on each chip.

[0133] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0134] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0135] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0136] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0137] 5) Remove the serum, add the primary antibody Anti-PR (ready-to-use), and store in a refrigerator at 4°C overnight;

[0138] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0139] 7) Add enzyme-labeled polymer secondary antibody and incubate in a 37°C incubator for 30 minutes. Prepare a negative control tissue chip under the same conditions.

[0140] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0141] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0142] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0143] 11) Conventional dehydration and transparency, neutral gum sealing.

[0144] 12) Observe the test film: hold the label side in your left hand and read the film from left to right and from top to bottom; the target cells are colored and located in the cell nucleus. The results are specific, clear and contrasting.

[0145] 3 (conventional method), Anti-Her2 antibody was provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Homemade breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slide (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0146] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0147] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0148] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0149] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0150] 5) Remove the serum, add the primary antibody Anti-Her2 (ready-to-use), and store in a refrigerator at 4°C overnight;

[0151] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0152] 7) Add enzyme-labeled polymer secondary antibody and incubate in a 37°C incubator for 30 minutes. Prepare a negative control tissue chip under the same conditions.

[0153] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0154] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloration;

[0155] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0156] 11) Conventional dehydration and transparency, neutral gum sealing.

[0157] 12) Observe the test slide: hold the label side in your left hand and read the slide from left to right and from top to bottom; the target cells are stained and located on the cell membrane, and the results are specific, clear and contrasting.

[0158] 4 (direct shaking method), Anti-Her2, Anti-ER, Anti-PR antibodies were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Homemade breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0159] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0160] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0161] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0162] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0163] 5) Remove the serum, directly add 500ul of the same mass and proportion of Anti-ER and Anti-Her2 into the antibody sampling tube, use a vortex mixer to mix, hold the slide in the left hand, double-scoop the tissue microarray slide, add Anti-PR and Anti-Her2 to cover the left half of the tissue microarray chip, and add Anti-ER and Anti-Her2 to cover the right half, and put it in a 4°C refrigerator overnight;

[0164] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0165] 7) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 30 minutes, and prepare a negative control tissue chip under the same conditions;

[0166] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0167] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0168] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0169] 11) Conventional dehydration and transparency, neutral gum sealing.

[0170] 12) Observe the test slide: hold the label side in your left hand and read the slide from left to right and from top to bottom; the target cells are stained and located on the cell membrane, and the results are specific, clear and contrasting.

[0171] 5 (front and back drop mixing method), Anti-Her2, Anti-ER, Anti-PR antibodies were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Homemade breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slide (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0172] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0173] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0), boil at medium heat for 10 minutes, stop heating for 5 minutes, keep warm, then switch to medium-low heat for 5 minutes to retrieval antigen, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0174] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0175] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0176] 5) Remove serum, hold the slide with your left hand, scoop up the left half of the tissue microarray slide, first add Anti-Her2 to cover the microarray chip, then add Anti-PR within 10 seconds, shake the slide evenly; for the right half of the tissue microarray chip, first add Anti-Her2 to cover the microarray chip, then add Anti-ER within 10 seconds, shake the slide evenly, and put it in a 4°C refrigerator overnight;

[0177] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0178] 7) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 30 minutes, and prepare a negative control tissue chip under the same conditions;

[0179] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0180] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloration;

[0181] 10) Hematoxylin counterstaining, water washing, and differentiation followed by thorough water washing to blue;

[0182] 11) Conventional dehydration and transparency, neutral gum sealing.

[0183] 12) Observe the test slide: hold the label side in your left hand and read the slide from left to right and from top to bottom; the target cells are stained and located on the cell membrane, and the results are specific, clear and contrasting.

[0184] 6 (indirect method), Anti-Her2, Anti-ER, Anti-PR antibodies were provided by Suzhou Baidao Medical Technology Co., Ltd., clone number: 246G0D3. Homemade breast cancer and its matching adjacent tissue combination chip continuous sectioning, double slides (two continuous tissue wax slides were attached to each glass slide), product number: F048Br01, 48 breast cancer tissue points per chip.

[0185] 1) Bake the slides at 60℃ for 30 minutes and dewax them normally;

[0186] 2) Antigen retrieval: microwave retrieval with Tris-EDTA antigen retrieval buffer (pH 9.0) at medium heat for 10 minutes until boiling, then turn off the heat for 5 minutes to keep warm and then turn to medium-low heat for 5 minutes to retrieval antigens, cool to room temperature, and wash with phosphate buffered saline (PBS) for 5 minutes × 3 times;

[0187] 3) Block endogenous peroxidase with 3% H2O2-methanol at room temperature for 30 minutes, and wash with PBS for 5 minutes × 3 times;

[0188] 4) Add normal non-immune animal serum and incubate at room temperature for 10 minutes;

[0189] 5) Remove the serum, hold the slide with your left hand, scoop up the left and right tissue microarray chips on the tissue chip slide, and add the primary antibody Anti-Her2 at the same time, and place it in a 4°C refrigerator overnight;

[0190] 6) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0191] 7) Add enzyme-labeled polymer secondary antibody and incubate in a 37°C incubator for 30 minutes. Prepare a negative control tissue chip under the same conditions.

[0192] 8) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0193] 9) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0194] 10) Hold the slide with your left hand, and add the primary antibodies Anti-PR and Anti-ER to the left and right tissue microarray chips on the tissue microarray slide at the same time; incubate at 37°C for 1 hour.

[0195] 11) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0196] 12) Add enzyme-labeled polymer secondary antibody to both left and right tissues at the same time, incubate in a 37°C incubator for 15 minutes, and prepare a negative control tissue chip under the same conditions;

[0197] 13) Wash with 0.1% Tween-20 PBS for 5 minutes × 3 times;

[0198] 14) DAB color development for 5 minutes, the positive expression area turns brown, and the color development is terminated by washing with distilled water; at the same time, observe the negative control: no coloring

[0199] Anti-ER, Anti-PR, and Anti-Her2 immunohistochemistry interpretation criteria (refer to the 2019 version of the Guidelines for Immunohistochemistry Testing of Breast Cancer): At least two senior pathologists in this field observe more than 100 invasive cancer cells;

[0200] Her2 interpretation criteria:

[0201] 0: no staining or ≤10% of infiltrating cancer cells showing incomplete and weak cell membrane staining;

[0202] 1+: >10% of infiltrating cancer cells show incomplete, weak cell membrane staining;

[0203] 2+: >10% of the infiltrating cancer cells showed weak to moderate intensity of intact cell membranes with brown-yellow staining or <30% of the infiltrating cancer cells showed strong and intact cell membranes with brown-brown staining;

[0204] 3+:>Infiltrating cancer cells show strong and complete brown staining of cell membrane.

[0205] ER and PR interpretation criteria:

[0206] -: <1% tumor cell nuclear staining in the presence of positive internal control and negative control;

[0207] +: ≥1% of tumor cells have brown or tan nuclei.

[0208] Consistency and reliability of interpretation of tissue microarray tissue points: The interpretation results of two senior pathologists on the test slides of this experiment were compared. The kappa calculation results were between 0 and 1, and can be divided into five groups to represent different levels of consistency:

[0209] 0.0-0.20 means very low consistency, 0.21-0.40 means fair consistency, 0.41-0.60 means moderate consistency, 0.61-0.80 means high consistency and 0.81-1 means almost perfect consistency.

[0210] The Kappa value of the judgment results of the two doctors in this experiment was 0.97, which showed that the consistency was quite reliable.

[0211] Results: The detection of ER, PR, Her-2 by cocktail-like immunohistochemistry double-label single staining method using breast cancer tissue microarray was compared with conventional section immunohistochemistry (the results were basically the same):

[0212] 1) The results of the two groups were compared and statistically analyzed. The chi-square test results of each group showed that P>0.5 (see Table 1 and Table 2)

[0213] Table 1 Expression of ER and PR in breast cancer immunohistochemistry tissue microarray and paraffin sections

[0214] method Antibody Name - + ++ +++ Organization chip points Chip direct shake method ER 20 4 6 16 46 Paraffin section conventional method PR 18 7 5 16 46

[0215] Note: x2=0.29, P>0.5, no significant difference

[0216] Table 2 Expression of Her-2 in breast cancer immunohistochemistry on tissue microarray and paraffin sections

[0217] method Antibody Name 0 1+ 2+ 3+ Organization Points Direct Shake Method Her-2 24 6 5 11 46 Paraffin section conventional method Her-2 24 6 3 13 46

[0218] Note: x2=0.05, P>0.5, no significant difference

[0219] As shown in Table 1, there is no significant difference in the results, which proves that the data of each group are highly consistent. Cocktail immunohistochemistry double labeling single staining method is a reliable immunohistochemistry method.

[0220] 2) The direct shaking method requires 1 tissue chip and 9 steps to complete the experiment (ER, PR, and Her-2 detection), the front-back mixing method requires 1 tissue chip and 12 steps to complete the experiment (ER, PR, and Her-2 detection), and the indirect method requires 1 tissue chip and 14 steps to complete the experiment (ER, PR, and Her-2 detection); while the conventional method requires 3 tissue chips and 27 steps to complete the experiment (ER, PR, and Her-2 detection).

[0221] 3) The test results were interpreted by senior pathologists in this field, and the kappa value of the results was 0.97, which proved that the detection of ER, PR, Her-2 by cocktail-like immunohistochemistry double-label single staining method of breast cancer tissue chip and conventional section immunohistochemistry detection had strong consistency and high credibility.

[0222] in conclusion:

[0223] The results of the cocktail-like immunohistochemistry double-label single staining method for detecting ER, PR, and Her-2 using breast cancer tissue chips are consistent and reliable with those of conventional immunohistochemistry sections. It is suitable for the detection of ER, PR, and Her-2 in breast cancer.

[0224] This method can simultaneously label and locate two different antibodies on a tissue chip, and does not require multiple color developers. It is simple, economical and reliable.

[0225] The experimental steps of direct shaking method are less than those of front-to-back mixing method, less than those of indirect method, and less than those of conventional method. Therefore, the direct shaking method of cocktail-like immunohistochemistry double antibody single label has the least experimental steps, but it requires mixing equipment such as vortex mixers. If such equipment is not available, the front-to-back mixing method is a better choice. Although the experimental steps of the indirect method are slightly increased, it provides space for the immunohistochemistry enzyme labeling method with 3 or more labels.

[0226] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A cocktail immunohistochemical detection method, characterized in that: The following steps are involved: S1. Dewaxing, antigen repairing and blocking the tissue sections to be examined in sequence; S2, adding more than 2 antibodies; S3, adding enzyme-labeled polymer secondary antibody, incubating and then washing; S4, add DAB color developer, and wash with water after color development; S5, add hematoxylin, wash with water until blue returns after differentiation, and complete immunohistochemical detection; The tissue section to be examined is divided into a left half and a right half A plurality of tissue points to be inspected are respectively arranged on the left half and the right half of the tissue section to be inspected; In S2, the adding of two or more antibodies specifically comprises the following steps: Add mixed antibody 1 to the left half of the tissue section to be tested, and add mixed antibody 2 to the right half of the tissue section to be tested, incubate and then wash; The mixed antibody 1 and the mixed antibody 2 respectively contain more than two kinds of antibodies; The types of antibodies in the mixed antibody 1 and the mixed antibody 2 are not completely the same.

2. The cocktail immunohistochemical detection method according to claim 1, characterized in that: The mixed antibody 1 or the mixed antibody 2 is obtained by vortex mixing the respective antibodies in equal proportions.

3. The cocktail immunohistochemical detection method according to claim 2, characterized in that: The mixed antibody 1 contains Anti-ER and Anti-Her2; The mixed antibody 2 contains Anti-PR and Anti-Her2.

4. The cocktail immunohistochemical detection method according to claim 1, characterized in that: In S2, the adding of two or more antibodies specifically comprises the following steps: Add antibody 1 to the left half of the tissue section to be tested, and add antibody 2 to the left half of the tissue section to be tested within 10 seconds and shake well; Add antibody 1 to the right half of the tissue section to be tested, and add antibody 3 to the right half of the tissue section to be tested within 10 seconds and shake well, incubate and wash; The antibody 1, antibody 2 and antibody 3 are antibodies against different target proteins.

5. The cocktail immunohistochemical detection method according to claim 4, characterized in that: The antibody 1 is Anti-Her2; The antibody 2 is Anti-PR; The antibody 3 is Anti-ER.

6. A cocktail immunohistochemical detection method, characterized in that: The following steps are involved: S1. Dewaxing, antigen repairing and blocking the tissue sections to be examined in sequence; S2, adding antibody 1 dropwise to the left and right halves of the tissue section to be tested, incubating and then washing; S3, adding enzyme-labeled polymer secondary antibody, incubating and then washing; S4, add DAB color developer, and wash with water after color development; S5, adding antibody 2 to the left half of the tissue section to be tested, and adding antibody 3 to the right half of the tissue section to be tested, incubating and then washing; S6, adding enzyme-labeled polymer secondary antibody, incubating and then washing; S7. Add DAB colorimetric agent, wash with water after color development, and complete the immunohistochemical detection.

7. The cocktail immunohistochemical detection method according to claim 6, characterized in that: The antibody 1 is Anti-Her2; The antibody 2 is Anti-PR; The antibody 3 is Anti-ER.

8. The cocktail immunohistochemical detection method according to claim 1 or 6, characterized in that: The tissue slice to be tested is a microarray tissue chip or a common tissue slice containing a solid phase carrier; The solid phase carrier is a glass slide.

9. The cocktail immunohistochemical detection method according to claim 8, characterized in that: The slide is an anti-shedding immunohistochemistry slide, an immunofluorescence in situ hybridization slide or a poly-lysine slide.

10. The cocktail immunohistochemical detection method according to claim 1 or 6, characterized in that: The dewaxing process is carried out by baking the sheet, the temperature of the baking sheet is 50-70°C, and the baking time is 20-40 minutes; And / or, the reagent used for antigen retrieval is Tris-EDTA antigen retrieval buffer; and / or, the pH of the Tris-EDTA antigen retrieval buffer is 8.0 to 9.0; And / or, the sealing process comprises the following steps: First, add H2O2-methanol to block endogenous peroxidase, incubate for 20-40 minutes, wash, add serum, and incubate for 5-15 minutes; and / or, the concentration of H2O2-methanol blocking endogenous peroxidase is 2-4%; And / or, the washing adopts Tween-20 PBS solution with a volume concentration of 0.05-0.2%.

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