Immunohistochemical double-staining color development system, detection kit and immunohistochemical double-staining detection method

Abstract

The invention relates to an immunohistochemical double-staining color development system, a detection kit and an immunohistochemical double-staining detection method, and belongs to the technical field of biomedicine. The immunohistochemical color development system is composed of HRP-Magenta, HRP-Cyan, HRP-Gold, AP-Gold and basic fuchsin dyeing reagents, the reagents are combined to form two color development systems including a sequential double-dyeing color development system and a combined double-dyeing color development system, dyeing in a sequential double-dyeing mode and a combined double-dyeing mode can be achieved, dipositive cells can present a third color, and the color development efficiency is improved. And two single positive cells can be obviously distinguished, so that the interpretation is more accurate and quicker. In addition, the sequential double-staining developing system is composed of the HRP system and the AP system, so that the selectivity of the whole developing system is higher, the requirements of different users can be met, meanwhile, the developing system can be customized according to the requirements of the users, and the use flexibility of the immunohistochemical double-staining reagent is improved.

Description

Technical Field

[0001] The invention relates to an immunohistochemical double staining color development system, a detection kit and an immunohistochemical double staining detection method, and belongs to the technical field of biomedicine. Background Art

[0002] Immunohistochemistry (IHC) is a staining technique used to detect specific proteins in tissues or cells. It uses antibodies and antigens to find the location of the required protein. However, in order to find two indicator protein expressions or structures in the same tissue, or to observe different cell morphologies in the same tissue, double staining technology is relatively important. Double staining technology can locate and detect two antigens in the same sample. The biggest difference from the fluorescence method is that there is no need to merge the pictures. The difference in the color of the two proteins can be found by the naked eye, and it can be preserved permanently without worrying about signal degradation. It not only saves the number of samples and preparation time, but also has a relatively low detection cost.

[0003] At present, the double staining technology on the market uses horseradish peroxidase (HRP) or alkaline phosphatase (AP) color development systems to color tissues. However, in the clinical detection process, it was found that the use of traditional DAB and Fast Red color development has the problem of unclear contrast and difficulty in distinguishing, especially the poor contrast between single positive cells (brown or red) and double positive cells. The reason for this linearity is that single positive cells are brown or red, and double positive cells are a mixture of brown and red. According to the different antibody expression levels, the mixed color will tend to be red or brown, which makes the distinction between double positive cells and single positive cells worse, and ultimately easy to misread, resulting in misdiagnosis. In addition, in scientific research, it is sometimes necessary to observe the interaction or co-localization position relationship of two proteins in the sample. The problem of poor color contrast of DAB and Fast Red in the traditional double staining technology will also affect the operator's inability to accurately judge the interaction or co-localization position relationship of the two proteins in the sample. Therefore, it is an urgent problem to develop a double staining color development system that can clearly distinguish the two single positive indicators and double positive indicators. Summary of the invention

[0004] The first object of the present invention is to provide an immunohistochemical double staining color development system to solve the problem that the color development system used for immunohistochemical double staining in the prior art cannot effectively distinguish different detection targets.

[0005] The second object of the present invention is to provide an immunohistochemical double staining detection kit to solve the problem that the color development system used for immunohistochemical double staining in the prior art cannot effectively distinguish different detection targets.

[0006] The third object of the present invention is to provide an immunohistochemical double staining detection method, which provides an immunohistochemical double staining detection method that can clearly distinguish between single positive indicators and double positive indicators.

[0007] In order to achieve the above object, the technical solution of an immunohistochemical double staining color development system of the present invention is: An immunohistochemical double staining color development system, the color development system includes a sequential double staining color development system and / or a combined double staining color development system; The color development system includes a sequential double-dye color development system and / or a combined double-dye color development system; The sequential double-staining color development system is an HRP system or an AP system; the HRP system selects two of HRP-Gold, HRP-Magenta, and HRP-Cyan; the AP system selects AP-Gold and basic fuchsin; The combined double-staining color development system is a combination of HRP-Cyan and AP-Gold, a combination of HRP-Magenta and AP-Gold, a combination of HRP-Gold and basic fuchsin, or a combination of HRP-Cyan and basic fuchsin.

[0008] The beneficial effect of the above scheme is that the immunohistochemical double staining color development system of the present invention is a pioneering invention. The color development system of the present invention includes a sequential double staining color development system and a combined double staining color development system; wherein the sequential double staining color development system is suitable for immunohistochemical sequential double staining, and the combined double staining color development system is suitable for immunohistochemical combined double staining.

[0009] Specifically, the HRP system in the sequential double staining color development system uses HRP-Magenta (SD8019), HRP-Gold (SD8016) and HRP-Cyan (SD8018). HRP-Magenta is colored red on the corresponding single positive cells, HRP-Gold is colored golden yellow on the corresponding single positive cells, HRP-Cyan is colored cyan on the corresponding single positive cells, HRP-Gold and HRP-Cyan will be colored together on the double positive cells, superimposed to form a third color green, HRP-Magenta and HRP-Cyan will be colored together, superimposed to form a third color blue, HRP-Magenta and HRP-Gold will be colored together, superimposed to form a third color orange. The AP system uses basic fuchsin staining reagent (SD8006) and AP-Gold (SD8017). The basic fuchsin staining reagent is colored red on the corresponding single positive cells, and AP-Gold is colored golden yellow on the corresponding single positive cells. On the double positive cells, basic fuchsin and AP-Gold will be colored together, superimposed to form a third color orange. The three colors have a good contrast, and the interpretation is fast and accurate. There is no restriction on the species of the target protein. It can be a mouse antibody or a rabbit antibody. It has the advantages of high staining specificity, easy optimization of conditions, and high flexibility.

[0010] The combined double-staining color development system uses HRP-Cyan (SD8018) and AP-Gold (SD8017). HRP-Cyan is colored cyan on the corresponding single-positive cells, and AP-Gold is colored golden yellow on the corresponding single-positive cells. On double-positive cells, HRP-Cyan and AP-Gold will be colored together, superimposed to form a third color of green. HRP-Magenta (SD8019) and AP-Gold can also be used. HRP-Magenta is colored red on the corresponding single-positive cells, and AP-Gold is colored golden yellow on the corresponding single-positive cells. On double-positive cells, HRP-Magenta and AP-Gold will be colored together, superimposed to form a third color of orange. HRP-Gold and basic fuchsin staining reagents can also be used. HRP-Gold is colored golden yellow on the corresponding single positive cells, and basic fuchsin staining reagents are colored red on the corresponding single positive cells. On double positive cells, HRP-Gold and basic fuchsin will be colored together, superimposed to form a third color orange. HRP-Cyan and basic fuchsin staining reagents can also be used. HRP-Cyan is colored cyan on the corresponding single positive cells, and basic fuchsin staining reagents are colored red on the corresponding single positive cells. On double positive cells, HRP-Cyan and basic fuchsin will be colored together, superimposed to form a third color blue. The three colors have a good contrast, and the interpretation is quick and accurate. The target protein needs to have one mouse antibody and one rabbit antibody, which has the advantages of simple operation, quick detection, and a very short overall detection process.

[0011] In order to achieve the above object, the technical scheme of an immunohistochemical double staining detection kit of the present invention is: An immunohistochemical double staining detection kit comprises the immunohistochemical double staining color development system.

[0012] The beneficial effect of the above scheme is that the present invention prepares the above immunohistochemical double staining color development system into the form of a kit, which, on the one hand, retains the advantages of the immunohistochemical double staining color development system, and on the other hand, is convenient for users to use, can provide users with a variety of choices, and can also customize the kit according to the needs of users to meet diversified market needs.

[0013] As a further improvement, the detection kit is an immunohistochemistry sequential double staining kit; the immunohistochemistry sequential double staining kit includes a first target primary antibody, a second target primary antibody, and a HRP or AP labeled secondary antibody.

[0014] As a further improvement, the detection kit includes a blocking agent.

[0015] As a further improvement, the detection kit is an immunohistochemistry combination double staining kit; the immunohistochemistry combination double staining kit includes a combination of primary antibodies and a combination of secondary antibodies for the target to be detected; the combination of secondary antibodies includes secondary antibodies labeled with AP and HRP, respectively; the antibody species of different targets in the combination of primary antibodies in the detection kit are different.

[0016] As a further improvement, the detection kit includes antigen repair solution, endogenous peroxidase blocking agent, and sealing agent.

[0017] As a further improvement, the targets are p53 and Ki-67, CK8 and CK18, P16 and Ki-67.

[0018] In order to achieve the above object, the technical solution of an immunohistochemical double staining detection method of the present invention is: An immunohistochemical double staining detection method, the immunohistochemical double staining detection method comprising sequential double staining and combined double staining; The sequential double staining process mainly includes section dewaxing and hydration, antigen repair, blocking, incubation with the first target primary antibody, incubation with the HRP-labeled secondary antibody, color development with the first color developing solution, incubation with a blocking agent, incubation with the second target primary antibody, incubation with the HRP-labeled secondary antibody, color development with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are selected from two of HRP-Gold, HRP-Magenta, and HRP-Cyan; Or the sequential double staining process mainly includes section dewaxing and hydration, antigen repair, incubation with the first target primary antibody, incubation with the AP-labeled secondary antibody, color development with the first color developing solution, incubation with a blocking agent, incubation with the second target primary antibody, incubation with the AP-labeled secondary antibody, color development with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are cross-selected from basic fuchsin and AP-Gold; The combined double staining process includes dewaxing and hydrating the sections, antigen repairing, blocking, incubating the combined primary antibodies of the targets to be detected, incubating the combined secondary antibodies, developing with the first color developing solution, developing with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are cross-selected from HRP-Cyan and AP-Gold, HRP-Magenta and AP-Gold, HRP-Gold and basic fuchsin, or HRP-Cyan and basic fuchsin.

[0019] The beneficial effect of the above scheme is that the immunohistochemical double staining detection method of the present invention uses the immunohistochemical double staining color development system, which can realize immunohistochemical sequential double staining and immunohistochemical combined double staining, wherein immunohistochemical sequential double staining has the advantages of high staining specificity, easy optimization of conditions, and high flexibility; immunohistochemical combined double staining has the advantages of simple operation, fast detection, and a very short overall detection process. Both immunohistochemical double staining modes can make the single positive index and the double positive index develop into different colors, so that the three indicators can be quickly and accurately distinguished.

[0020] Furthermore, the immunohistochemical double staining detection method provided by the present invention can realize automatic staining on a fully automatic immunohistochemical staining machine, whether it is sequential immunohistochemical double staining or combined immunohistochemical double staining, which is convenient and fast, stable and reproducible, and has a wide range of audiences, and can be used for clinical testing, scientific research, and other life science fields. Moreover, the two immunohistochemical double staining modes each have their own advantages, and users can choose according to actual needs, and the audience range is wide.

[0021] As a further improvement, the targets are p53 and Ki-67, CK8 and CK18, P16 and Ki-67. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Example 19 of the present invention (100×); Figure 2 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Example 20 of the present invention (100×); Figure 3 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Example 21 of the present invention (100×); Figure 4 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Example 22 of the present invention (100×); Figure 5 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Example 23 of the present invention (100×); Figure 6 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Example 24 of the present invention (100×); Figure 7 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Example 25 of the present invention (100×); Figure 8 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Example 26 of the present invention (100×); Fig. 9 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Example 27 of the present invention (100×); Fig.10 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Example 28 of the present invention (100×); Fig.11 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 29 of the present invention (100×); Fig.12 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 30 of the present invention (100×); Fig.13 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 31 of the present invention (100×); Fig.14 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 32 of the present invention (100×); Fig.15 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 33 of the present invention (100×); Fig.16 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Example 34 of the present invention (100×); Fig.17 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Comparative Example 1 of the present invention (100×); Fig.18 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Comparative Example 2 of the present invention (100×); Fig.19 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Comparative Example 3 of the present invention (100×); Fig. 20 The results of p53 / Ki-67 immunohistochemical double staining of esophageal tissue in Comparative Example 4 of the present invention (100×); Fig.21 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Comparative Example 5 of the present invention (100×); Fig. 22 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Comparative Example 6 of the present invention (100×); Fig.23 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Comparative Example 7 of the present invention (100×); Fig.24 The results of CK8 / CK18 immunohistochemical double staining of breast tissue in Comparative Example 8 of the present invention (100×); Fig.25 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Comparative Example 9 of the present invention (100×); Fig.26 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Comparative Example 10 of the present invention (100×); Fig. 27The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in Comparative Example 11 of the present invention (100×); Fig.28 The results of p16 / Ki-67 immunohistochemical double staining of cervical tissue in comparative example 12 of the present invention (100×). DETAILED DESCRIPTION

[0023] The present invention has found through a large number of preliminary investigations that the color development systems DAB staining solution and Fast Red staining solution commonly used in immunohistochemical double staining cannot effectively distinguish single positive targets from double positive targets after color development; and through a large number of experiments, it has been found that the color contrast before and after the superposition of the color staining solution currently available on the market is poor after immunohistochemical color development, which is also likely to cause the problem of being unable to effectively distinguish single positive targets from double positive targets. Based on this, the present invention provides an immunohistochemical double staining color development system.

[0024] The immunohistochemical color development system of the present invention is composed of HRP-Magenta (red), HRP-Cyan (cyan), HRP-Gold (golden yellow), AP-Gold (golden yellow), and alkaline fuchsin staining reagent (red). The above reagents are combined to form two color development systems, namely, a sequential double staining color development system and a combined double staining color development system. The sequential double staining and combined double staining mode staining can be achieved, and both can make double positive cells present a third color, which can be clearly distinguished from two single positive cells, making the interpretation more accurate and faster. Moreover, the sequential double staining color development system is composed of an HRP system and an AP system, which makes the entire color development system more selective, which is conducive to meeting the needs of different users. At the same time, the color development system can also be customized according to the needs of users, thereby improving the flexibility of using immunohistochemical double staining reagents.

[0025] The present invention is further described in detail below in conjunction with specific examples. Unless otherwise specified, the equipment and reagents used in each embodiment, experimental example and comparative example can be obtained from commercial sources.

[0026] The following examples and comparative examples use a fully automatic immunohistochemical staining instrument produced by Henan Synotech Biotechnology Co., Ltd., model: CNT330.

[0027] The color developing solution used in the examples of the present invention was purchased from Henan Synotech Biotechnology Co., Ltd. The specific product number and registration number are as follows: HRP-Gold colorimetric solution, product number SD8016, registration number Yuzhengxiebei20240262; HRP-Cyan colorimetric solution, product number SD8018, registration number Yuzhengxiebei20240263; HRP-Magenta colorimetric solution, product number SD8019, registration number Yuzhengxiebei20240261; AP-Gold colorimetric solution, product number SD8017, registration number Yuzhengxiebei20240264; Basic fuchsin color developing reagent color developing solution, product number is SD8006, registration number is Yuzhengxiebei20200186.

[0028] 1. A specific embodiment of an immunohistochemical double staining color development system of the present invention: Example 1 The immunohistochemical double staining color development system of this embodiment is a sequential double staining color development system; the sequential double staining color development system is an HRP system; the HRP system consists of a first color development liquid and a second color development liquid; the first color development liquid is HRP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8016; the second color development liquid is HRP-Cyan, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8018.

[0029] Example 2 The immunohistochemical double staining color development system of this embodiment is a combined double staining color development system; the combined double staining color development system consists of a first color development liquid and a second color development liquid; the first color development liquid is basic fuchsin, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8006; the second color development liquid is HRP-Cyan, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8018.

[0030] Example 3 The immunohistochemical double staining color development system of this embodiment is a combined double staining color development system; the combined double staining color development system consists of a first color development liquid and a second color development liquid; the first color development liquid is HRP-Magenta, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8019; the second color development liquid is AP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8017.

[0031] Example 4 The immunohistochemical double staining color development system of this embodiment is a sequential double staining color development system; the sequential double staining color development system is an HRP system; the HRP system consists of a first color development liquid and a second color development liquid; the first color development liquid is HRP-Magenta, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8019; the second color development liquid is HRP-Cyan, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8018.

[0032] Example 5 The immunohistochemical double staining color development system of this embodiment is a sequential double staining color development system; the sequential double staining color development system is an HRP system; the HRP system consists of a first color development liquid and a second color development liquid; the first color development liquid is HRP-Magenta, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8019; the second color development liquid is HRP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8016.

[0033] Example 6 The immunohistochemical double staining color development system of this embodiment is a sequential double staining color development system; the sequential double staining color development system is an AP system; the AP system consists of a first color development liquid and a second color development liquid; the first color development liquid is basic fuchsin, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8006; the second color development liquid is AP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8017.

[0034] Example 7 The immunohistochemical double staining color development system of this embodiment is a combined double staining color development system; the combined double staining color development system consists of a first color development liquid and a second color development liquid; the first color development liquid is AP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8017; the second color development liquid is HRP-Cyan, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8018.

[0035] Example 8 The immunohistochemical double staining color development system of this embodiment is a combined double staining color development system; the combined double staining color development system consists of a first color development liquid and a second color development liquid; the first color development liquid is basic fuchsin, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8006; the second color development liquid is HRP-Gold, purchased from Henan Synotech Biotechnology Co., Ltd., with the item number SD8016.

[0036] 2. Specific embodiments of an immunohistochemical double staining detection kit of the present invention: Example 9 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting p53 and Ki-67, wherein antibody A is p53, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CPM-0140, and antibody B is Ki-67, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CKM-0032; (4) The secondary antibody reagent was HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number RS4001-2; (5) The first colorimetric solution was HRP-Gold colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8016; (6) The second color developing solution is HRP-Cyan color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (8) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (9) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (10) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0037] Example 10 (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting p53 and Ki-67, wherein antibody A is p53, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CPM-0140, and antibody B is Ki-67, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CKM-0032; (4) The secondary antibody reagent was HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number RS4001-2; (5) The first color developing solution was HRP-Magenta color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8019; (6) The second color developing solution is HRP-Cyan color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (8) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (9) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (10) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0038] Embodiment 11 (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting p53 and Ki-67, wherein antibody A is p53, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CPM-0140, and antibody B is Ki-67, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CKM-0032; (4) The secondary antibody reagent was HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number RS4001-2; (5) The first color developing solution was HRP-Magenta, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8019; (6) The second color developing solution is HRP-Gold color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8016; (7) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (8) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (9) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (10) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0039] Example 12 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting CK18 and CK8, wherein antibody A is CK18, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1093, and antibody B is CK8, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1004; (4) The secondary antibody reagent was HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number RS4001-2; (5) The first colorimetric solution was HRP-Gold colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8016; (6) The second colorimetric solution was HRP-Cyan colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (8) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (9) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (10) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0040] Example 13 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting CK18 and CK8, wherein antibody A is CK18, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1093, and antibody B is CK8, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1004; (4) The secondary antibody reagent was HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number RS4001-2; (5) The first color developing solution was HRP-Magenta color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8019; (6) The second color developing solution is HRP-Cyan color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (8) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (9) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (10) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0041] Embodiment 14 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Primary antibody reagent: The primary antibody reagent includes antibody A and antibody B for detecting CK18 and CK8, wherein antibody A is CK18, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1093, and antibody B is CK8, purchased from Henan Synotech Biotechnology Co., Ltd., with a catalog number of CCM-1004; (3) The secondary antibody reagent was AP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8003; (4) The first color developing solution is alkaline fuchsin color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8006; (5) The second colorimetric solution was AP-Gold colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8017; (6) The primary antibody reagent was rabbit anti-mouse polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3206-1; (7) Blocker, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8010; (8) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (9) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0042] Embodiment 15 (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) p16 / Ki-67 combined primary antibody: The combined primary antibody is a mixed antibody mainly composed of antibody A for detecting p16 and antibody B for detecting Ki67. Antibody A is a mouse anti-p16 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CPM-0104. Antibody B is a rabbit anti-Ki-67 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CKR-0034. (4) HRP & AP combined secondary antibody: The combined secondary antibody is a mixed antibody mainly composed of AP enzyme-labeled goat anti-mouse polyclonal antibody and HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8002-2; (5) The first color developing solution was AP-Gold staining reagent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8017; (6) The second colorimetric solution was HRP-Cyan colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (8) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0043] Example 16 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) p16 / Ki-67 combined primary antibody: The combined primary antibody is a mixed antibody mainly composed of antibody A for detecting p16 and antibody B for detecting Ki67. Antibody A is a mouse anti-p16 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CPM-0104. Antibody B is a rabbit anti-Ki-67 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CKR-0034. (4) HRP & AP combined secondary antibody: The combined secondary antibody is a mixed antibody mainly composed of AP enzyme-labeled goat anti-mouse polyclonal antibody and HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8002-2; (5) The first color developing solution is basic fuchsin staining reagent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8006; (6) The second colorimetric solution was HRP-Cyan colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8018; (7) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (8) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0044] Embodiment 17 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) p16 / Ki-67 combined primary antibody: The combined primary antibody is a mixed antibody mainly composed of antibody A for detecting p16 and antibody B for detecting Ki67. Antibody A is a mouse anti-p16 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CPM-0104. Antibody B is a rabbit anti-Ki-67 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CKR-0034. (4) HRP & AP combined secondary antibody: The combined secondary antibody is a mixed antibody mainly composed of AP enzyme-labeled goat anti-mouse polyclonal antibody and HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8002-2; (5) The first color developing solution was HRP-Magenta color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8019; (6) The second colorimetric solution was AP-Gold colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8017; (7) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (8) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0045] Embodiment 18 The immunohistochemical double staining detection kit of this embodiment includes the following components: (1) Immunohistochemistry antigen retrieval buffer (EDTA method), catalog number SD2003; (2) Peroxidase blocking agent, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD3101-1; (3) p16 / Ki-67 combined primary antibody: The combined primary antibody is a mixed antibody mainly composed of antibody A for detecting p16 and antibody B for detecting Ki67. Antibody A is a mouse anti-p16 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CPM-0104. Antibody B is a rabbit anti-Ki-67 monoclonal antibody purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number CKR-0034. (4) HRP & AP combined secondary antibody: The combined secondary antibody is a mixed antibody mainly composed of AP enzyme-labeled goat anti-mouse polyclonal antibody and HRP enzyme-labeled goat anti-rabbit polyclonal antibody, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8002-2; (5) The first color developing solution is alkaline fuchsin color developing solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8006; (6) The second colorimetric solution was HRP-Gold colorimetric solution, purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8016; (7) Sealing medium was purchased from Henan Synotech Biotechnology Co., Ltd., catalog number SD8008; (8) Cleaning solution: PBST buffer (pH 7.4) or TBST buffer (pH 8.0).

[0046] 3. Specific embodiment of an immunohistochemical double staining detection method of the present invention: Embodiment 19 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 9 to perform p53 / Ki-67 tissue double staining in sequence on esophageal tissue samples, wherein p53 is developed using HRP-Gold and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as follows: 1. Dewaxing and hydration The paraffin sections were placed in fresh xylene and soaked twice for 10 min each time; after removing excess liquid, they were placed in anhydrous ethanol and soaked twice for 5 min each time; after removing excess liquid, they were placed in 95% ethanol and soaked for 5 min; after removing excess liquid, they were placed in 85% ethanol and soaked for 5 min; after removing excess liquid, they were placed in 70% ethanol and soaked for 5 min; and rinsed with purified water for 5 min.

[0047] 2. Antigen retrieval Heat the antigen retrieval buffer in a pressure cooker on an induction cooker until boiling; place the dewaxed and hydrated sections on a high-temperature staining rack and put them into the pressure cooker; cover the lid and continue heating until steam is emitted, start timing for 2.5 minutes, then remove the pressure cooker from the heat source and let it cool naturally for 5 minutes; rinse with tap water to cool it, remove the valve and open the lid, and take out the sections after the liquid in the pot cools naturally to room temperature; soak them in purified water twice, 3 minutes each time; soak them in cleaning solution once, 3 minutes each time.

[0048] 3. Add endogenous peroxidase blocking agent After the immunohistochemistry pen draws a circle around the tissue, rinse it with purified water, and then soak it in the cleaning solution for 3 minutes. Remove the cleaning solution, add 100 μL of endogenous peroxidase blocking agent to the tissue area to be tested, and incubate it at room temperature for 5 minutes. Then soak it in the cleaning solution twice, 5 minutes each time.

[0049] 4. Incubation with primary antibody A The washing solution was removed, and 100 μL of p53 primary antibody reagent was added, incubated at 37°C for 30 min, and soaked in the washing solution twice, 5 min each time.

[0050] 5. Primary Antibody and Reagent Incubation Remove the cleaning solution, add 100 μL of primary antibody reagent, incubate at 37°C for 30 min, and soak in the cleaning solution twice, 5 min each time.

[0051] 6. Secondary Antibody Incubation Remove the cleaning solution, add 100 μL of secondary antibody reagent, incubate at 37°C for 20 min, and soak in the cleaning solution twice, 5 min each time.

[0052] 7. HRP-Gold color development Remove the cleaning solution, add 100 μL of freshly prepared HRP-Gold, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0053] 8. Blocking Remove the deionized water, add 100 μL of blocking agent, incubate at 70°C for 15 min, and soak in the cleaning solution twice, 5 min each time.

[0054] 9. Primary Antibody B Incubation The washing solution was removed, and 100 μL of Ki-67 primary antibody reagent was added dropwise, incubated at 37°C for 30 min, and soaked in the washing solution twice, 5 min each time.

[0055] 10. Primary Antibody and Reagent Incubation Remove the cleaning solution, add 100 μL of primary antibody reagent, incubate at 37°C for 10 min, and soak in the cleaning solution twice, 5 min each time.

[0056] 11. Secondary Antibody Incubation Remove the cleaning solution, add 100 μL of secondary antibody reagent, incubate at 37°C for 20 min, and soak in the cleaning solution twice, 5 min each time.

[0057] 12. HRP-Cyan color development Remove the cleaning solution, add 100 μL of freshly prepared HRP-Cyan, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0058] 13. Seal the film Add 100 μL of aqueous mounting medium to completely cover the tissue, dry at 50-60°C, and then add neutral gum to mount the slice.

[0059] Observe the staining results as Figure 1 As shown in the figure, it can be seen that in the two groups of samples on the left and right, the p53 single-positive cells appear golden yellow, the Ki-67 single-positive cells appear cyan, and the p53 / Ki-67 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0060] Embodiment 20 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 10 to perform p53 / Ki-67 tissue double staining in sequence on esophageal tissue samples, wherein p53 is developed using HRP-Magenta staining reagent and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as described in Example 19.

[0061] Observe the staining results as Figure 2 As shown in the figure, it can be seen that p53 single-positive cells appear red, Ki-67 single-positive cells appear cyan, and p53 / Ki-67 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0062] Embodiment 21 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 11 to perform p53 / Ki-67 tissue double staining in sequence on esophageal tissue samples, wherein p53 is developed using HRP-Magenta and Ki-67 is developed using HRP-Gold. The specific implementation operation is as described in Example 19.

[0063] Observe the staining results as Figure 3 As shown in the figure, it can be seen that p53 single-positive cells appear red, Ki-67 single-positive cells appear yellow, and p53 / Ki-67 double-positive cells appear orange. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0064] Embodiment 22 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 9, and uses a fully automatic immunohistochemical staining machine to perform p53 / Ki-67 tissue double staining on the esophageal tissue sample, wherein p53 is developed using HRP-Gold and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as follows: The automatic immunohistochemical staining machine performs sequential double staining procedures including the following steps: 1. Dewaxing and hydration of paraffin tissue sections; 2. Antigen repair: Use antigen repair buffer to repair the slices under high pressure at 100°C for 20 minutes with a sample volume of 150 μL; 3. Add 3%~3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150μL of sample; 4. The first step is to incubate the p53 primary antibody: incubate at 37°C for 30 minutes, with a sample volume of 150 μL; 5. Incubation after the first antibody in the first procedure: incubate at 37°C for 10 minutes, with a sample volume of 150 μL; 6. Incubation of the first procedure secondary antibody: incubate at 37°C for 10 minutes, with a sample volume of 150 μL; 7. HRP-Gold color development: incubate at room temperature for 30-40 minutes, with a sample volume of 150 μL; 8. Blocking agent: 70℃ for 10 minutes, sample volume is 150μL; 9. Second procedure: Ki-67 primary antibody incubation: incubate at 37°C for 30 minutes, with a sample volume of 150 μL; 10. Second procedure: incubate at 37°C for 10 minutes, with a sample volume of 150 μL. 11. Second procedure: incubate with secondary antibody at 37°C for 10 minutes, with a sample volume of 150 μL; 12. HRP-Cyan color development: incubate at room temperature for 30-40 minutes, with a sample volume of 150 μL; 13. Seal the slides with water-based sealing glue.

[0065] Observe the staining results as Figure 4 As shown in the figure, it can be seen that p53 single-positive cells appear golden yellow, Ki-67 single-positive cells appear cyan, and p53 / Ki-67 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0066] Embodiment 23 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 10, and uses a fully automatic immunohistochemical staining machine to perform p53 / Ki-67 tissue double staining on the esophageal tissue sample, wherein p53 is developed using HRP-Magenta staining reagent and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as described in Example 22: Observe the staining results as Figure 5 As shown in the figure, it can be seen that p53 single-positive cells appear red, Ki-67 single-positive cells appear cyan, and p53 / Ki-67 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0067] Embodiment 24 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 12 to perform CK8 / CK18 tissue double staining in sequence on breast tissue samples, wherein CK18 is developed using HRP-Gold and CK8 is developed using HRP-Cyan. The specific implementation operation is as described in Example 19.

[0068] Observe the staining results as Figure 6 As shown in the figure, it can be seen that CK18 single-positive cells appear golden yellow, CK8 single-positive cells appear cyan, and CK8 / CK18 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0069] Embodiment 25 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 13 to perform CK8 / CK18 tissue double staining in sequence on breast tissue samples, wherein CK18 is developed using HRP-Magenta staining reagent and CK8 is developed using HRP-Cyan. The specific implementation operation is as described in Example 19.

[0070] Observe the staining results as Figure 7 As shown in the figure, it can be seen that CK18 single-positive cells appear red, CK8 single-positive cells appear cyan, and CK8 / CK18 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0071] Embodiment 26 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 14 to perform CK8 / CK18 tissue combination double staining on breast tissue samples, wherein CK18 is developed using basic fuchsin and CK8 is developed using AP-Gold. The specific implementation operation is described as follows.

[0072] 1. Dewaxing and hydration The paraffin sections were placed in fresh xylene and soaked twice for 10 min each time; after removing excess liquid, they were placed in anhydrous ethanol and soaked twice for 5 min each time; after removing excess liquid, they were placed in 95% ethanol and soaked for 5 min; after removing excess liquid, they were placed in 85% ethanol and soaked for 5 min; after removing excess liquid, they were placed in 70% ethanol and soaked for 5 min; and rinsed with purified water for 5 min.

[0073] 2. Antigen retrieval Heat the antigen retrieval buffer in a pressure cooker on an induction cooker until boiling; place the dewaxed and hydrated sections on a high-temperature staining rack and put them into the pressure cooker; cover the lid and continue heating until steam is emitted, start timing for 2.5 minutes, then remove the pressure cooker from the heat source and let it cool naturally for 5 minutes; rinse with tap water to cool it, remove the valve and open the lid, and take out the sections after the liquid in the pot cools naturally to room temperature; soak them in purified water twice, 3 minutes each time; soak them in cleaning solution once, 3 minutes each time.

[0074] 3. Primary Antibody A Incubation After the immunohistochemistry pen draws a circle around the tissue, rinse it with purified water, then soak it in cleaning solution for 3 minutes, remove the cleaning solution, add 100 μL of CK18 primary antibody reagent, incubate at 37°C for 30 minutes, and soak it in the cleaning solution twice, 5 minutes each time.

[0075] 4. Primary Antibody and Reagent Incubation Remove the cleaning solution, add 100 μL of primary antibody reagent, incubate at 37°C for 30 min, and soak in the cleaning solution twice, 5 min each time.

[0076] 5. Secondary Antibody Incubation Remove the cleaning solution, add 100 μL of secondary antibody reagent, incubate at 37°C for 20 min, and soak in the cleaning solution twice, 5 min each time.

[0077] 6. Basic fuchsin color development Remove the cleaning solution, add 100 μL of freshly prepared basic fuchsin, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0078] 7. Blocking Remove the deionized water, add 100 μL of blocking agent, incubate at 70°C for 15 min, and soak in the cleaning solution twice, 5 min each time.

[0079] 8. Primary Antibody B Incubation The cleaning solution was removed, and 100 μL of CK8 primary antibody reagent was added, incubated at 37°C for 30 min, and soaked in the cleaning solution twice, 5 min each time.

[0080] 9. Primary Antibody and Reagent Incubation Remove the cleaning solution, add 100 μL of primary antibody reagent, incubate at 37°C for 10 min, and soak in the cleaning solution twice, 5 min each time.

[0081] 10. Secondary Antibody Incubation Remove the cleaning solution, add 100 μL of secondary antibody reagent, incubate at 37°C for 20 min, and soak in the cleaning solution twice, 5 min each time.

[0082] 11. AP-Gold color development Remove the cleaning solution, add 100 μL of freshly prepared AP-Gold, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0083] 12. Seal the film Add 100 μL of aqueous mounting medium to completely cover the tissue, dry at 50-60°C, and then add neutral gum to mount the slice.

[0084] Observe the staining results as Figure 8 As shown in the figure, it can be seen that CK18 single-positive cells appear red, CK8 single-positive cells appear yellow, and CK8 / CK18 double-positive cells appear orange. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0085] Embodiment 27 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 12, and uses a fully automatic immunohistochemical staining machine to perform CK8 / 18 tissue sequential double staining on breast tissue samples, wherein CK18 is developed using HRP-Gold and CK8 is developed using HRP-Cyan. The specific implementation operation is as described in Example 22.

[0086] Observe the staining results as Fig. 9 As shown in the figure, it can be seen that CK18 single-positive cells appear golden yellow, CK8 single-positive cells appear cyan, and CK8 / CK18 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0087] Embodiment 28 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 13, and uses a fully automatic immunohistochemical staining machine to perform CK8 / CK18 tissue double staining in sequence on breast tissue samples, wherein CK18 is developed using HRP-Magenta staining reagent and CK8 is developed using HRP-Cyan. The specific implementation operation is as described in Example 22.

[0088] Observe the staining results as Fig.10As shown in the figure, it can be seen that CK18 single-positive cells appear red, CK8 single-positive cells appear cyan, and CK8 / CK18 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0089] Embodiment 29 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 15 to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using AP-Gold colorimetric reagent and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as follows: 1. Dewaxing and hydration The paraffin sections were placed in fresh xylene and soaked twice for 15 minutes each time; after removing the excess liquid, they were placed in anhydrous ethanol and soaked twice for 5 minutes each time; after removing the excess liquid, they were placed in 95% ethanol and soaked for 5 minutes; after removing the excess liquid, they were placed in 85% ethanol and soaked for 5 minutes; after removing the excess liquid, they were placed in 70% ethanol and soaked for 5 minutes; and rinsed with purified water for 5 minutes.

[0090] 2. Antigen retrieval Heat the antigen retrieval buffer in a pressure cooker on an induction cooker until it boils; place the dewaxed and hydrated sections on a high-temperature staining rack. Put it in the pressure cooker; cover the lid and continue heating until steam is emitted. After starting the timing for 2.5 minutes, remove the pressure cooker from the heat source and cool it naturally for 5 minutes; rinse it with tap water to cool it, remove the valve and open the lid, wait for the liquid in the pot to cool naturally to room temperature, then take out the slices; soak it in purified water twice, 3 minutes each time; soak it once with cleaning solution, 3 minutes each time.

[0091] 3. Add endogenous peroxidase blocking agent After the immunohistochemistry pen draws a circle around the tissue, rinse it with purified water, and then soak it in the cleaning solution for 3 minutes. Remove the cleaning solution, add 100 μL of endogenous peroxidase blocking agent to the tissue area to be tested, and incubate it at room temperature for 5 minutes. Then soak it in the cleaning solution twice, 5 minutes each time.

[0092] 4. p16 / Ki67 combined primary antibody incubation The washing solution was removed, and 100 μL of p16 / Ki67 combined primary antibody reagent was added, incubated at 37°C for 60 min, and soaked in the washing solution twice, 5 min each time.

[0093] 5. HRP&AP combined secondary antibody incubation Remove the cleaning solution, add 100 μL of HRP&AP combined secondary antibody reagent, incubate at 37°C for 30 min, and soak in the cleaning solution twice, 5 min each time.

[0094] 6. AP-Gold color development solution Remove the cleaning solution, add 100 μL of freshly prepared AP-Gold colorimetric solution, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0095] 7. HRP-Cyan staining solution Remove the cleaning solution, add 100 μL of freshly prepared HRP-Cyan staining solution, incubate at 37°C for 30-40 min, and soak in deionized water twice, 5 min each time.

[0096] 8. Seal the film Add 100 μL of aqueous mounting medium to completely cover the tissue, dry at 50-60°C, and then add neutral gum to mount the slice.

[0097] Observe the staining results as Fig.11 As shown in the figure, it can be seen that p16 single-positive cells appear golden yellow, Ki-67 single-positive cells appear cyan, and p16 / Ki-67 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0098] Embodiment 30 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 16 to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using basic fuchsin staining reagent and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as described in Example 29.

[0099] Observe the staining results as Fig.12 As shown in the figure, it can be seen that p16 single-positive cells appear red, Ki-67 single-positive cells appear cyan, and p16 / Ki-67 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0100] Embodiment 31 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 17 to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using HRP-Magenta and Ki-67 is developed using AP-Gold. The specific implementation operation is as described in Example 29.

[0101] Observe the staining results as Fig.13 As shown in the figure, it can be seen that p16 single-positive cells appear red, Ki-67 single-positive cells appear yellow, and p16 / Ki-67 double-positive cells appear orange. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0102] Embodiment 32 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 18 to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using basic fuchsin and Ki-67 is developed using HRP-Gold. The specific implementation operation is as described in Example 29.

[0103] Observe the staining results as Fig.14 As shown in the figure, it can be seen that p16 single-positive cells appear red, Ki-67 single-positive cells appear yellow, and p16 / Ki-67 double-positive cells appear orange. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0104] Embodiment 33 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 15, and uses a fully automatic immunohistochemical staining machine to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using an AP-Gold staining kit and Ki-67 is developed using HRP-Cyan. The specific implementation operation is described as follows.

[0105] The combined double staining procedure of the fully automatic immunohistochemical staining machine includes the following steps: 1. Dewaxing and hydration of paraffin tissue sections; 2. Antigen repair: Use antigen repair buffer to repair the slices under high pressure at 100°C for 20 minutes with a sample volume of 150 μL; 3. Add 3% to 3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150 μL of sample.

[0106] 4. Incubation of p16 / Ki67 combination primary antibody: incubate at 37°C for 60 minutes, with a sample volume of 150 μL; 5. Incubation of HRP&AP combination secondary antibody: incubate at 37°C for 30 minutes, with a sample volume of 150 μL; 6. AP-Gold color development: incubate at room temperature for 30-40 minutes, with a sample volume of 150 μL; 7. HRP-Cyan color development: incubate at room temperature for 30-40 minutes, with a sample volume of 150 μL; 8. Seal the slides with water-based sealing glue.

[0107] Observe the staining results as Fig.15 As shown in the figure, it can be seen that p16 single-positive cells appear golden, Ki-67 single-positive cells appear cyan, and p16 / Ki-67 double-positive cells appear green. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0108] Embodiment 34 An immunohistochemical double staining detection method of this embodiment uses the immunohistochemical double staining detection kit of Example 16, and uses a fully automatic immunohistochemical staining machine to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 is developed using a basic fuchsin staining kit and Ki-67 is developed using HRP-Cyan. The specific implementation operation is as described in Example 33.

[0109] Observe the staining results as Fig.16 As shown in the figure, it can be seen that p16 single-positive cells appear red, Ki-67 single-positive cells appear cyan, and p16 / Ki-67 double-positive cells appear blue. The contrast between the three types of cells is very clear and strong, and it is easy to see at a glance.

[0110] 4. Comparative Proportion Comparative Example 1 In this comparative example, p53 / Ki-67 tissue double staining was performed on esophageal tissue samples, wherein p53 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; Ki-67 was developed using DAB, purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number SD3001. The specific operating steps are as described in Example 19.

[0111] Observe the staining results as Fig.17 As can be seen from the figure, p53 single-positive cells appear red, Ki-67 single-positive cells appear brown, and p53 / Ki-67 double-positive cells appear a mixture of red and brown. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0112] Comparative Example 2 In this comparative example, p53 / Ki-67 tissue double staining was performed on esophageal tissue samples, wherein p53 used StayYellow / HRP, purchased from Abcam, with the catalog number ab169561; Ki-67 was developed using HRP-Blue, purchased from Bio SB, with the catalog number BSB 0145. The specific operating steps are as described in Example 19.

[0113] Observe the staining results as Fig.18 As can be seen from the figure, p53 single-positive cells appear yellow, Ki-67 single-positive cells appear blue, and p53 / Ki-67 double-positive cells appear a mixture of yellow and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0114] Comparative Example 3 In this comparative example, p53 / Ki-67 tissue double staining was performed on esophageal tissue samples, wherein p53 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; Ki-67 was developed using HRP-Blue, purchased from Bio SB, with the catalog number BSB 0145. The specific operation steps are as described in Example 19.

[0115] Observe the staining results as Fig.19 As can be seen from the figure, p53 single-positive cells appear red, Ki-67 single-positive cells appear blue, and p53 / Ki-67 double-positive cells appear a mixture of red and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0116] Comparative Example 4 In this comparative example, p53 / Ki-67 tissue double staining was performed on esophageal tissue samples, wherein p53 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; Ki-67 was developed using StayYellow / HRP, purchased from Abcam, with the catalog number ab169561. The specific operation steps are as described in Example 19.

[0117] Observe the staining results as Fig. 20 As can be seen from the figure, p53 single-positive cells appear red, Ki-67 single-positive cells appear yellow, and p53 / Ki-67 double-positive cells appear a mixture of red and yellow. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0118] Comparative Example 5 In this comparative example, CK8 / CK18 tissue double staining was performed on breast tissue samples, wherein CK18 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; CK8 was developed using DAB, purchased from Henan Synotech Biotechnology Co., Ltd., with the catalog number SD3001. The specific operating steps are as described in Example 19.

[0119] Observe the staining results as Fig.21 As can be seen from the figure, CK18 single-positive cells appear red, CK8 single-positive cells appear brown, and CK8 / CK18 double-positive cells appear a mixture of red and brown. The contrast between the three types of cells is poor and it is difficult to distinguish them.

[0120] Comparative Example 6 In this comparative example, CK8 / CK18 tissue double staining was performed on breast tissue samples, wherein CK18 was developed using StayYellow / HRP, purchased from Abcam, with the catalog number ab169561; CK8 was developed using HRP-Blue, purchased from BioSB, with the catalog number BSB 0145. The specific operation steps are as described in Example 19.

[0121] Observe the staining results as Fig. 22 As can be seen from the figure, CK18 single-positive cells appear yellow, CK8 single-positive cells appear blue, and CK8 / CK18 double-positive cells appear a mixture of yellow and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0122] Comparative Example 7 In this comparative example, CK8 / CK18 tissue combination double staining was performed on breast tissue samples, wherein CK18 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; CK8 was developed using HRP-Blue, purchased from Bio SB, with the catalog number BSB 0145. The specific operation steps are as described in Example 19.

[0123] Observe the staining results as Fig.23 As can be seen from the figure, CK18 single-positive cells appear red, CK8 single-positive cells appear blue, and CK8 / CK18 double-positive cells appear a mixture of red and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0124] Comparative Example 8 In this comparative example, CK8 / CK18 tissue double staining was performed on breast tissue samples, wherein CK18 was developed using AlkMagenta, purchased from Bio SB, with the catalog number BSB 0081; CK8 was developed using StayYellow / HRP, purchased from Abcam, with the catalog number ab169561. The specific operation steps are as described in Example 19.

[0125] Observe the staining results as Fig.24 As can be seen from the figure, CK18 single-positive cells appear red, CK8 single-positive cells appear yellow, and CK8 / CK18 double-positive cells appear a mixture of red and yellow. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0126] Comparative Example 9 In this comparative example, p16 / Ki-67 tissue combination double staining was performed on cervical tissue samples, wherein Ki-67 was developed using AlkMagenta, purchased from Bio SB, with the product number BSB 0081; p16 was developed using DAB, purchased from Henan Synotech Biotechnology Co., Ltd., with the product number SD3001. The specific operating steps are as described in Example 29.

[0127] Observe the staining results as Fig.25 As can be seen from the figure, p16 single-positive cells appear brown, Ki-67 single-positive cells appear red, and p16 / Ki-67 double-positive cells appear a mixture of red and brown. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0128] Comparative Example 10 In this comparative example, p16 / Ki-67 tissue double staining was performed on cervical tissue samples, wherein p16 was developed using StayYellow / HRP, purchased from Abcam, with the catalog number ab169561; Ki-67 was developed using HRP-Blue, purchased from BioSB, with the catalog number BSB 0145. The specific operation steps are as described in Example 19.

[0129] Observe the staining results as Fig.26 As can be seen from the figure, p16 single-positive cells appear yellow, Ki-67 single-positive cells appear blue, and p16 / Ki-67 double-positive cells appear a mixture of yellow and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0130] Comparative Example 11 In this comparative example, p16 / Ki-67 tissue combination double staining was performed on cervical tissue samples, wherein p16 was developed using AlkMagenta, purchased from Bio SB, with the product number BSB 0081; Ki-67 was developed using HRP-Blue, purchased from Bio SB, with the product number BSB 0145. The specific operating steps are as described in Example 29.

[0131] Observe the staining results as Fig. 27 As can be seen from the figure, p16 single-positive cells appear red, Ki-67 single-positive cells appear blue, and p16 / Ki-67 double-positive cells appear a mixture of red and blue. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0132] Comparative Example 12 In this comparative example, a fully automatic immunohistochemical staining machine was used to perform p16 / Ki-67 tissue combination double staining on cervical tissue samples, wherein p16 was developed using Alk Magenta, purchased from Bio SB, with the catalog number BSB0081; Ki-67 was developed using StayYellow / HRP, purchased from Abcam, with the catalog number ab169561. The specific operating steps are as described in Example 33.

[0133] Observe the staining results as Fig.28 As can be seen from the figure, p16 single-positive cells appear red, Ki-67 single-positive cells appear yellow, and p16 / Ki-67 double-positive cells appear a mixture of red and yellow. The contrast between the three types of cells is poor, and it is difficult to distinguish them.

[0134] The above description is only an illustrative embodiment of the present invention and is not intended to limit the scope of the present invention. Any equivalent changes and modifications made by a person of ordinary skill in the art without departing from the concept and principle of the present invention shall fall within the scope of protection of the present invention.

Claims

1. An immunohistochemical double staining color development system, characterized in that: The color development system includes a sequential double-dye color development system and / or a combined double-dye color development system; The sequential double-staining color development system is an HRP system or an AP system; the HRP system selects two of HRP-Gold, HRP-Magenta, and HRP-Cyan; the AP system selects AP-Gold and basic fuchsin; The combined double-staining color development system is a combination of HRP-Cyan and AP-Gold, a combination of HRP-Magenta and AP-Gold, a combination of HRP-Gold and basic fuchsin, or a combination of HRP-Cyan and basic fuchsin.

2. An immunohistochemical double staining detection kit, characterized in that: The detection kit comprises the immunohistochemical double staining color development system according to claim 1.

3. The immunohistochemical double staining detection kit according to claim 2, characterized in that: The detection kit is an immunohistochemical sequential double staining kit; the immunohistochemical sequential double staining kit comprises a first target primary antibody, a second target primary antibody, and a HRP or AP labeled secondary antibody.

4. The immunohistochemical double staining detection kit according to claim 3, characterized in that: The detection kit includes a blocking agent.

5. The immunohistochemical double staining detection kit according to claim 2, characterized in that: The detection kit is an immunohistochemistry combined double staining kit; the immunohistochemistry combined double staining kit includes a combined primary antibody and a combined secondary antibody for the target to be detected; the combined secondary antibody includes secondary antibodies labeled with AP and HRP, respectively; the antibody species of different targets in the combined primary antibody of the detection kit are different.

6. The immunohistochemical double staining detection kit according to any one of claims 3 to 5, characterized in that: The detection kit comprises an antigen repair solution, an endogenous peroxidase blocking agent and a sealing medium.

7. The immunohistochemical double staining detection kit according to claim 6, characterized in that: The targets are p53 and Ki-67, CK8 and CK18, P16 and Ki-67.

8. An immunohistochemical double staining detection method, characterized in that: The immunohistochemical double staining detection method includes sequential double staining and combined double staining; The sequential double staining process mainly includes section dewaxing and hydration, antigen repair, blocking, incubation with the first target primary antibody, incubation with the HRP-labeled secondary antibody, color development with the first color developing solution, incubation with a blocking agent, incubation with the second target primary antibody, incubation with the HRP-labeled secondary antibody, color development with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are selected from two of HRP-Gold, HRP-Magenta, and HRP-Cyan; Or the sequential double staining process mainly includes section dewaxing and hydration, antigen repair, incubation with the first target primary antibody, incubation with the AP-labeled secondary antibody, color development with the first color developing solution, incubation with a blocking agent, incubation with the second target primary antibody, incubation with the AP-labeled secondary antibody, color development with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are cross-selected from basic fuchsin and AP-Gold; The combined double staining process includes dewaxing and hydrating the sections, antigen repairing, blocking, incubating the combined primary antibodies of the targets to be detected, incubating the combined secondary antibodies, developing with the first color developing solution, developing with the second color developing solution, and sealing; the first color developing solution and the second color developing solution are cross-selected from HRP-Cyan and AP-Gold, HRP-Magenta and AP-Gold, HRP-Gold and basic fuchsin, or HRP-Cyan and basic fuchsin.

9. The immunohistochemical double staining detection method according to claim 8, characterized in that: The targets are p53 and Ki-67, CK8 and CK18, P16 and Ki-67.

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