Immunohistochemical multi-staining section as well as preparation method and application thereof

Abstract

The invention discloses an immunohistochemical multi-staining section as well as a preparation method and application thereof, and belongs to the technical field of pathological detection. The multi-staining section is provided with an epithelium-derived malignant tumor staining labeled antibody and a staining labeled antibody of an infringed tissue, the epithelium-derived malignant tumor staining labeled antibody is an AE1 / AE3 antibody, and the staining labeled antibody of the infringed tissue is one or more of an S-100 antibody, a CD31 antibody, a CD34 antibody or an NSE antibody. The section is provided with the overlapped dyeing marks, on one hand, the problem that the requirement for the number of sample sections is high is solved, on the other hand, the invasion relation between epithelium-derived malignant tumor components and nerves and blood vessels can be directly and clearly displayed on one section, and the requirement for accurate diagnosis of pathologists is met.

Description

Technical Field

[0001] The present invention relates to the technical field of pathological detection, and particularly to an immunohistochemical multiple staining section and a preparation method and application thereof. Background Art

[0002] In clinical pathological detection, radical specimens of malignant tumors often need to be evaluated for nerve and blood vessel invasion, which is used as a basis for guiding subsequent clinical treatment and prognosis evaluation of patients.

[0003] Immunohistochemistry is a practical and effective examination method in pathological diagnosis. Through the specific binding of antigens and antibodies, qualitative and localization studies of proteins in tissues or cells can be achieved, and it can be used for the diagnosis, classification, treatment guidance and prognosis judgment of tumors. With the development of this technology and the emergence of various specific antibodies, its practical value in tumor diagnosis and differentiation has been widely recognized.

[0004] However, each tumor tissue section can only be used to label one immunohistochemical antibody. In actual work, there are mainly two problems:

[0005] 1) Single staining is limited by the size of pathological tissue lesions. A single tumor tissue needs to be combined with multiple antibodies to be clear. When the components of the lesions to be observed and differentiated are less, such as when the observed lesion area is extremely small and the number of sections is limited, it is difficult to meet the diagnostic requirements for the sample quantity required for immunohistochemistry.

[0006] 2) Currently, there are many antibodies used to evaluate the invasion of nerves and blood vessels in epithelial-derived malignant tumors, and separate staining is required. After each of multiple consecutive sections is stained separately, the invasion relationship between tumor tissues and nerves and blood vessels is difficult to be clearly shown on one section at a glance, and it is necessary to comprehensively judge by memorizing and repeatedly comparing multiple sections. Moreover, the pathological tissues of consecutive different sections will change, resulting in difficulty in accurately positioning the invasion of tumor cells, nerves and blood vessels, and it is difficult to meet the accurate diagnosis needs of pathologists. Summary of the Invention

[0007] Aiming at the problem of the lack of immunohistochemical multiple staining for epithelial-derived malignant tumors in the above-mentioned prior art, the present invention provides an immunohistochemical multiple staining section with superimposed markings, which on the one hand solves the problem of high demand for the number of sample sections, and on the other hand can directly and clearly show the invasion relationship between epithelial-derived malignant tumor components and nerves and blood vessels on one section, meeting the accurate diagnosis needs of pathologists.

[0008] The present invention discloses an immunohistochemical multiple staining section, on which there are staining marker antibodies for malignant tumors of epithelial origin and staining marker antibodies for invaded tissues. The staining marker for the malignant tumor of epithelial origin is the AE1 / AE3 antibody, and the staining marker for the invaded tissue is one or more of the S-100 antibody, CD31 antibody, CD34 antibody or NSE antibody.

[0009] In order to solve the problem in the conventional technology that it is difficult to visually interpret the invasion relationship between tumor tissues and blood vessels and nerves on a single section, the inventor considered using immunohistochemical overlay staining (double staining) method. By using immunohistochemistry technology, two or more antibodies are simultaneously labeled on the same tissue section, and the two antibodies are distinguished by different colors, so as to accurately evaluate their qualitative, locational, morphological and functional interrelationships.

[0010] In the selection of antibodies, after investigation and research, considering that the broad-spectrum keratin AE1 / AE3 is widely present in epithelial tissues and is an important marker for tumors of epithelial origin, using it as the marker for tumor tissues of epithelial origin, positive cytoplasmic staining in brown can be observed in conventional immunohistochemical single staining. When further selecting antibody markers for nerve tissues or vascular tissues, the specificity and sensitivity of the antibodies are compared through experiments, and then factors such as cost performance are comprehensively considered. Finally, it is confirmed that the S-100 antibody or NSE antibody is used to label nerve tissues, and the CD31 antibody or CD34 antibody is used to label vascular structures.

[0011] Among them, AE1 / AE3 is expressed in the cytoplasm of epithelial-derived cells, S-100 is expressed in the nucleus and cytoplasm of nerve tissues, NSE is expressed in the cytoplasm of nerve tissues, and CD31 and CD34 are expressed in the cell membrane and cytoplasm of vascular endothelial cells. Through multiple staining, the tumor tissue is stained with AE1 / AE3 (such as DAB color development, with brown being positive), and the normal nerve tissue is stained with S-100 or NSE (such as RED color development, with red being positive) to explore whether malignant tumor cells invade nerve tissues. The normal blood vessels are stained with CD31 or CD34 (such as RED color development, with red being positive) to explore whether malignant tumor cells invade blood vessels.

[0012] In some of the schemes, the invaded tissues are nerves and blood vessels, the staining marker antibody for the nerves is the S-100 antibody, and the staining marker antibody for the blood vessels is the CD31 antibody.

[0013] In some of the schemes, the AE1 / AE3 antibody is a mouse antibody, and the S-100 antibody and CD31 antibody are rabbit antibodies.

[0014] On the other hand, the present invention discloses a method for preparing the above-mentioned immunohistochemical multiplex staining section, which includes the following multiplex staining steps: taking a tissue section, dropping the staining labeled antibody for epithelial-derived malignant tumor and the staining labeled antibody for the invaded tissue, performing primary antibody incubation, then dropping the secondary antibodies respectively binding to the above-mentioned antibodies, performing secondary antibody incubation, adding chromogenic agents respectively targeting the above-mentioned secondary antibodies for chromogenic reaction, and then performing hematoxylin counterstaining treatment to obtain the section.

[0015] In some of the embodiments, in the multiplex staining step, the dosage ratio of the staining labeled antibody for epithelial-derived malignant tumor to the staining labeled antibody for the invaded tissue is 1:1.

[0016] In some of the embodiments, in the multiplex staining step, the staining labeled antibody for epithelial-derived malignant tumor and the staining labeled antibody for the invaded tissue are formulated as a mixed primary antibody solution for use, and the mixed primary antibody solution is a mixed working solution of AE1 / AE3 antibody, S-100 antibody and CD31 antibody with a dosage ratio of 1:1:1.

[0017] In some of the embodiments, in the multiplex staining step, the secondary antibodies are formulated as a mixed secondary antibody solution for use.

[0018] In some of the embodiments, in the multiplex staining step, the chromogenic agents are DAB and RED chromogenic solutions, and each chromogenic solution performs chromogenic reaction for 10 minutes respectively.

[0019] In some of the embodiments, in the multiplex staining step, the chromogenic agents are DAB and RED chromogenic solutions, and first RED chromogenic reaction is performed for 10 minutes, and then DAB chromogenic reaction is performed for 10 minutes.

[0020] In some of the embodiments, in the multiplex staining step, the hematoxylin counterstaining time is 5 minutes.

[0021] In some of the embodiments, in the multiplex staining step, the temperature of the primary antibody incubation is 27°C - 45°C, preferably 37°C.

[0022] In some of the embodiments, in the multiplex staining step, the time of the primary antibody incubation is 30 minutes.

[0023] In some of the embodiments, in the multiplex staining step, the temperature of the secondary antibody incubation is 27°C - 45°C, preferably 37°C.

[0024] In some of the embodiments, in the multiplex staining step, the time of the secondary antibody incubation is 20 minutes.

[0025] In some of these solutions, before the multiple staining step, there is also a pre-staining treatment step, and the pre-staining treatment step is as follows: Take the tissue to be stained, embed it, section it, bake the sections, and then dewax the tissue sections, perform antigen retrieval, and block peroxidase. It can be understood that the above specific steps can be carried out according to the conventional operations in the art.

[0026] In some of these solutions, after the multiple staining step, there is also a post-staining treatment step, and the post-staining treatment step is as follows: Dehydrate the stained sections and then mount them. It can be understood that the above specific steps can be carried out according to the conventional operations in the art.

[0027] On the other hand, the present invention also discloses a method for pathological interpretation of epithelial-derived malignant tumors for non-diagnostic and non-therapeutic purposes, using the above immunohistochemical multiple staining sections: In some of these solutions, when the staining marker antibody for the invaded tissue is the S-100 antibody or the NSE antibody, the immunohistochemical multiple staining sections are used to judge the invasion of epithelial-derived malignant tumors into nerves; when the staining marker antibody for the invaded tissue is the CD31 antibody or the CD34 antibody, the immunohistochemical multiple staining sections are used to judge the invasion of epithelial-derived malignant tumors into blood vessels; It can be understood that the above non-diagnostic and non-therapeutic purposes include scientific research, etc.

[0028] On the other hand, the present invention also discloses a kit for pathological interpretation of epithelial-derived malignant tumors, including the above staining marker antibodies for epithelial-derived malignant tumors, the above staining marker antibodies for the invaded tissue, as well as the secondary antibodies for the staining marker antibodies of epithelial-derived malignant tumors, the secondary antibodies for the staining marker antibodies of the invaded tissue, the chromogenic agent for the secondary antibody of the staining marker antibody of epithelial-derived malignant tumors, and the chromogenic agent for the secondary antibody of the staining marker antibody of the invaded tissue. Among them, the chromogenic agent for the secondary antibody of the staining marker antibody of epithelial-derived malignant tumors is the DAB chromogenic agent, and the chromogenic agent for the secondary antibody of the staining marker antibody for nerves or blood vessels is the RED chromogenic agent.

[0029] The above "AE1 / AE3" refers to broad-spectrum cytokeratin. The antibody AE1 / AE3 can react with cytokeratins CK1-8, 10, 14, 15, 16, 19, and is mainly used to label keratinized epithelium, stratified squamous epithelium, multilayer epithelium, proliferating keratinocytes, and simple epithelium, and is a broad-spectrum cytokeratin marker.

[0030] The above "S-100" refers to the S-100 protein, which is a low-molecular-weight soluble protein and is a glial marker.

[0031] The above "NSE" refers to neuron-specific enolase, which is a tumor marker for the diagnosis, prognosis, and follow-up of small cell lung cancer (SCLC), and also has certain value in the diagnosis, staging, and treatment of related neuroendocrine tumors (NETs).

[0032] The above-mentioned "CD31" refers to platelet endothelial cell adhesion molecule (Platelet endothelial cell adhesion molecule-1, PECAM-1 / CD31), which is expressed in most types of vascular tumors such as hemangioendothelioma, angiofibroma, hemangioma, and angiosarcoma.

[0033] The above-mentioned "CD34" refers to hematopoietic progenitor cell antigen, which is a transmembrane sialic acid glycoprotein with a molecular weight of 110 kDa. It is selectively expressed on the surface of human and other mammalian hematopoietic stem / progenitor cells and gradually weakens and disappears as the cells mature. It is also expressed in normal vascular endothelial cells, splenic marginal zone cells, and dendritic cells surrounding blood vessels, nerves, muscle fiber bundles, skin appendages, and the stroma of breast lobules. Most vascular tumors are positive. It is mainly used for the study of bone marrow hematopoietic diseases and various soft tissue diseases.

[0034] The above-mentioned "secondary antibody" refers to an antibody that can bind to an antibody, that is, an antibody against an antibody. Its main function is to detect the presence of an antibody (i.e., the primary antibody) and amplify the signal of the primary antibody. It can be understood that the secondary antibody is made by immunizing a heterologous animal using the property that an antibody is a macromolecular protein with antigenicity, and the immunoglobulin against this antibody is produced by the immune system of the heterologous animal. The secondary antibody is reactive to all antibodies (such as IgG, IgM, or IgA, etc.) of a specific species (such as mice or rabbits, etc.). For example, the secondary antibodies selected in the present invention are mouse anti- or rabbit anti- respectively to distinguish the staining marker antibodies of epithelial-derived malignant tumors and the staining marker antibodies of the invaded tissues.

[0035] The above-mentioned "DAB" is short for 3,3'-diaminobenzidine and is the core substrate in the chromogenic reaction of peroxidase. Under specific catalytic conditions, DAB can be converted into a water-insoluble brown product. This product not only has a distinct color and is easy to observe, but also has high stability and persistence, and is widely used in spot detection and the staining step in immunohistochemistry. Through the chromogenic reaction of DAB, the experimenter can visually observe the binding situation of the antigen and the antibody, so as to accurately judge the experimental results.

[0036] The above-mentioned "RED" is short for Fast-red (fast red chromogenic), which is a chromogenic agent widely used in immunohistochemical staining. It can react with alkaline phosphatase (AP) to produce a red precipitate, is insoluble in organic solvents, and can be covered with a permanent mounting medium.

[0037] On the basis of conforming to the common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0038] The reagents and raw materials used in the present invention are all commercially available.

[0039] The positive and progressive effects of the present invention are as follows:

[0040] For the immunohistochemical multiple staining sections of the present invention, the AE1 / AE3 antibody is used in combination with the S-100 antibody, CD31 antibody, CD34 antibody, or NSE antibody. Through the immunohistochemical double staining technique, it is used for marking the invasion relationship between malignant tumor cells of epithelial origin and nerves or blood vessels, helping doctors quickly and accurately assist in evaluating the invasion of nerves and blood vessels, and ultimately indicating the progression degree of malignant tumors of epithelial origin, bringing great convenience for clinicians to guide patients' postoperative treatment and evaluate prognosis.

[0041] Moreover, this multiple staining section can also solve the problem that when the pathological tissue lesion area is very small, multiple sections are required for single immunohistochemical staining, and the pathological tissue cannot be effectively utilized. And it reduces the consumption of consumables and the workload of pathologists in interpreting two sections through multiple staining. At the same time, it can also avoid the differences in tissue cells on two glass slides caused by tissue block trimming and multiple sectioning, further improving the accuracy of interpretation. Description of the Drawings

[0042] Figure 1 Photograph of AE1 / AE3 and S-100 multiple staining in Example 1.

[0043] Figure 2 Photograph of AE1 / AE3 and CD31 multiple staining in Example 1.

[0044] Figure 3 Photograph of AE1 / AE3 and NSE multiple staining in Example 1.

[0045] Figure 4 Photograph of AE1 / AE3 and CD34 multiple staining in Example 1.

[0046] Figure 5 Photographs of double antibody fusion staining of AE1 / AE3 rabbit / S100 mouse and AE1 / AE3 rabbit / CD31 mouse in Group a of Example 2. Among them, A is the staining section of AE1 / AE3 rabbit / S100 mouse, and B is the staining section of AE1 / AE3 rabbit / CD31 mouse.

[0047] Figure 6 Photographs of double antibody fusion staining of AE1 / AE3 rabbit / S100 mouse and AE1 / AE3 rabbit / CD31 mouse in Group b of Example 2. Among them, A is the staining section of AE1 / AE3 rabbit / S100 mouse, and B is the staining section of AE1 / AE3 rabbit / CD31 mouse.

[0048] Figure 7Photographs of group c of double antibody fusion staining of AE1 / AE3 rabbit / S100 mouse and AE1 / AE3 rabbit / CD31 mouse in Example 2. Among them, A is the stained section of AE1 / AE3 rabbit / S100 mouse, and B is the stained section of AE1 / AE3 rabbit / CD31 mouse.

[0049] Figure 8 Photographs of group d of double antibody fusion staining of AE1 / AE3 rabbit / S100 mouse and AE1 / AE3 rabbit / CD31 mouse in Example 2. Among them, A is the stained section of AE1 / AE3 rabbit / S100 mouse, and B is the stained section of AE1 / AE3 rabbit / CD31 mouse.

[0050] Figure 9 Photographs of group e of double antibody fusion staining of AE1 / AE3 rabbit / S100 mouse and AE1 / AE3 rabbit / CD31 mouse in Example 2. Among them, A is the stained section of AE1 / AE3 rabbit / S100 mouse, and B is the stained section of AE1 / AE3 rabbit / CD31 mouse.

[0051] Figure 10 Photographs of the section of multi - antibody fusion staining of AE1 / AE3 rabbit / S100 mouse / CD31 mouse in Example 2.

[0052] Figure 11 Photographs of the section of multi - antibody fusion staining of AE1 / AE3 rabbit / S100 mouse / CD31 mouse in Example 3.

[0053] Figure 12 Photographs of the section of AE1 / AE3 stained alone in Example 3.

[0054] Figure 13 Photographs of the section of S - 100 stained alone in Example 3.

[0055] Figure 14 Photographs of the section of CD31 stained alone in Example 3. Detailed implementation manners

[0056] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, such as hematoxylin staining, etc., they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0057] Primary antibody AE1 / AE3: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product number: ZM - 0069; Type: Mouse anti - human.

[0058] Primary antibody AE1 / AE3: Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., Product number: RAB - 0050; Type: Rabbit anti - human.

[0059] CD31 primary antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product Catalog Number: ZA-0568; Type: Rabbit anti-human.

[0060] CD31 primary antibody: Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., Product Catalog Number: MAB-0720; Type: Mouse anti-human.

[0061] S-100 primary antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product Catalog Number: ZM-0224; Type: Mouse anti-human.

[0062] S-100 primary antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product Catalog Number: ZA-0025; Type: Rabbit anti-human.

[0063] CD34 primary antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product Catalog Number: ZM-0046; Type: Mouse anti-human.

[0064] CD34 primary antibody: Manufacturer: Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Product Catalog Number: ZA-0550; Type: Rabbit anti-human.

[0065] NSE primary antibody: Manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., Product Catalog Number: kit-00014; Type: Mouse anti-human.

[0066] Mixed secondary antibody: Manufacturer: Leica Microsystems (Shanghai) Trading Co., Ltd., Product Catalog Number: DS9477

[0067] AP-Red chromogenic reagent: Manufacturer: Leica Microsystems (Shanghai) Trading Co., Ltd.; Product Catalog Number: DS9390.

[0068] DAB chromogenic reagent: Manufacturer: Leica Microsystems (Shanghai) Trading Co., Ltd.; Product Catalog Number: DS9800.

[0069] Example 1

[0070] This example screens the antibody types selected for multiplex staining.

[0071] 1. Method

[0072] According to different antibody combination methods, while keeping other experimental conditions unchanged, immunohistochemical staining was performed on 60 samples of epithelial-derived malignant tumors that needed to evaluate nerve and blood vessel invasion, and the multiplex staining (counterstaining) effects of AE1 / AE3 with S-100, CD31, CD34, and NSE were observed.

[0073] 1.1 Pretreatment before staining

[0074] ① Tissue section preparation:

[0075] Take malignant tumor tissues of epithelial origin, embed them and then section them. The specific method is as follows: First, trim the wax block before sectioning; then section the tissue continuously at 2 - 3 μm; after that, put the cut tissue sections into an oven at 55 - 65 °C and bake for 20 - 30 min.

[0076] ② Process after loading: Place the baked tissue sections on the staining channel of the BOND MAX Leica grouping machine. Set the program as follows:

[0077] First step, dewaxing: Drop 100 μl of dewaxing solution on the tissue sections, set the temperature at 72 °C, and the dewaxing time at 30 s. After dewaxing, wash with absolute ethanol 3 times, 1 min each time, and then rinse with deionized water.

[0078] Second step, repair: Drop 100 μl of repair solution on the tissue sections, set the temperature at 100 °C, and the repair time at 20 min. After repair, rinse with the washing solution 3 times, then drop peroxidase for blocking for 12 min, and rinse with the washing solution 3 times again to perform subsequent staining treatment.

[0079] 1.2 Staining

[0080] According to the antibodies and reagents selected in Table 1 below, perform staining with two different antibodies successively on one section. The preparation, dosage, etc. of the reagents used are carried out according to the reagent instructions. The specific operations are as follows:

[0081] Drop the first primary antibody working solution, incubate at an appropriate temperature (such as 37 °C) for a period of 30 min. After incubation, wash with the washing solution 2 - 3 times (30 s - 1 min each time), then drop the corresponding secondary antibody working solution, incubate at the same temperature for a period of time (20 min). After incubation, wash with the washing solution 2 - 3 times, drop the DAB chromogenic solution, and develop color for 5 min.

[0082] Drop the second primary antibody working solution, incubate at an appropriate temperature (such as 37 °C) for a period of 30 min. After incubation, wash with the washing solution 2 - 3 times (30 s - 1 min each time), then drop the corresponding secondary antibody working solution, incubate at the same temperature for a period of time (20 min). After incubation, wash with the washing solution 2 - 3 times again, drop the RED chromogenic solution, and develop color for 5 min.

[0083] After completing the above multiple staining, perform hematoxylin counterstaining, and stain with hematoxylin for 10 min.

[0084] 1.3 Dehydration, mounting and microscopic examination

[0085] For dehydration and mounting, routine methods can be used. For example, the glass slides can be placed in water and rinsed for 1 - 3 minutes, and then dehydrated and cleared by placing them in 80% ethanol for 3 minutes, 95% ethanol for 3 minutes, absolute ethanol for 3 minutes, absolute ethanol for 3 minutes, the first TO for 3 minutes, the second TO for 3 minutes, and the third TO for 3 minutes respectively, and finally mounted with neutral balsam.

[0086] Table 1. Multiple staining antibody combinations

[0087]

[0088] Note: "AE1 / AE3 is a broad-spectrum epithelial marker, which includes CK1-8, 10, 14-16 and 19, but does not include CK17 and CK18".

[0089] 2. Results

[0090] The staining results of the above groups are as Figure 1-4 shown, and the magnification is 100 times.

[0091] The multiple staining results of AE1 / AE3 and S-100 are as Figure 1 shown. It can be seen that the positive expression site of AE1 / AE3 is the cytoplasm, showing brownish-yellow; the positive expression site of S-100 is the nucleus, showing red.

[0092] The multiple staining results of AE1 / AE3 and CD31 are as Figure 2 shown. It can be seen that the positive expression site of AE1 / AE3 is the cytoplasm, showing brownish-yellow; the positive expression site of CD31 is the cytoplasm and cell membrane, showing red.

[0093] The multiple staining results of AE1 / AE3 and NSE are as Figure 3 shown. It can be seen that the positive expression site of AE1 / AE3 is the cytoplasm, showing brownish-yellow; the positive expression site of NSE is the cytoplasm, showing red.

[0094] The multiple staining results of AE1 / AE3 and CD34 are as Figure 4 shown. It can be seen that the positive expression site of AE1 / AE3 is the cytoplasm, showing brownish-yellow; the positive expression site of CD34 is the cytoplasm and cell membrane, showing red.

[0095] It can be seen from the above results that AE1 / AE3 superimposed with S-100 or NSE on the same section can efficiently determine whether there is nerve invasion in cancer cells, and AE1 / AE3 superimposed with CD31 or CD34 on the same section can efficiently determine whether there is vascular invasion in cancer cells.

[0096] Calculate the effective rates of each group and further evaluate the effective rates of each group. For the sections prepared by the method of this embodiment, the localization of the two antibodies under the microscope is accurate and the contrast is obvious. That is, the effective rate is the proportion of the samples that can be effectively interpreted by the diagnostician divided by the total number of samples. The effective rates of each group are shown in the following table.

[0097] Table 2. Effective Rates of Multiple Staining

[0098]

[0099] Among them, an invalid interpretation means that the preparation effect does not meet the requirements of diagnostic film reading, and a misdiagnosis means that a case with invasion is not diagnosed as having invasion or a case without invasion is diagnosed as having invasion.

[0100] The effective rate is calculated by the following method: (the number of specimens that can be effectively interpreted by the diagnostician / the total number of samples) × 100%

[0101] The accuracy rate is calculated by the following method: (the total number of samples - the number of samples with invalid interpretation - the number of misdiagnosed cases) / the total number of samples) × 100%

[0102] It can be seen from the above results that the effective rates of the superimposed color development preparations of AE1 / AE3 with S-100 or CD31 are relatively higher, the effect is the best, and the accuracy rate for evaluating nerve or vascular invasion in radical specimens of epithelial-derived malignant tumors is the highest.

[0103] Example 2

[0104] In this example, the staining method of the antibody combination selected in Example 1 is optimized.

[0105] 1. Method

[0106] According to different staining sequences, while keeping other experimental conditions unchanged, immunohistochemical staining was performed on the above 60 specimens respectively, and the multiple staining (double staining) effects of different staining processes were observed. The following is the verification experiment after preliminary screening.

[0107] 1.1 Pretreatment before staining

[0108] Same as Example 1.

[0109] 1.2 Antibody staining

[0110] (1) Sequential staining of double antibodies (AE1 / AE3 / S100, AE1 / AE3 / CD31):

[0111] Add the first primary antibody working solution (AE1 / AE3), incubate at an appropriate temperature (such as 37°C) for a certain period of time (such as 30 min), after incubation, rinse 2 - 3 times with the washing solution (30 s - 1 min each time), then add the corresponding secondary antibody working solution and incubate at the same temperature for a certain period of time (20 min). After incubation, rinse again 2 - 3 times with the washing solution, and add DAB for color development for 5 min.

[0112] Then add the second primary antibody working solution (S - 100, CD31), incubate at an appropriate temperature (such as 37°C) for a certain period of time (such as 30 min), after incubation, rinse 2 - 3 times with the washing solution (30 s - 1 min each time), then add the corresponding secondary antibody working solution and incubate at the same temperature for a certain period of time (such as 20 min). After incubation, rinse again 2 - 3 times with the washing solution, and add RED for color development for 5 min.

[0113] Subsequently, hematoxylin counterstaining, dehydration, mounting and microscopic examination were carried out according to the method of Example 1. The preparation effect is as Figure 1-2 .

[0114] (2) Double - antibody fusion staining (AE1 / AE3 rabbit / S100 mouse, AE1 / AE3 rabbit / CD31 mouse):

[0115] According to the principle of specific binding of antigen - antibody, cross - reactions are likely to occur when two or more antibodies are mixed. Therefore, during the experiment, the inventor first carried out antibody screening through the IHC staining step to determine whether the antibodies can be used in combination. After determining the antibody combination, adjustments were made. For example, multiple strict tests were carried out on the pH value of the repair solution, the concentration ratio and incubation time of the antibodies, the color development sequence and time of the color - developing solution, and the hematoxylin counterstaining time, and the best experimental procedure that can prevent two different antibodies from interfering with each other during co - staining was obtained. Part of the screening process is as follows.

[0116] Staining steps:

[0117] Add the mixed working solution of two primary antibodies (AE1 / AE3 rabbit anti - body and S100 mouse anti - body), incubate at an appropriate temperature (such as 37°C) for a period of 30 min, after incubation, rinse 2 - 3 times with the washing solution (30 s - 1 min each time), then add the mixed secondary antibody working solution and incubate at the same temperature for a certain period of time (20 min).

[0118] After incubation, rinse again 2 - 3 times with the washing solution, and add DAB and RED color - developing solutions respectively for color development for 5 min and 5 min in sequence.

[0119] Through preliminary experiments, the conditions were explored first, and combined with the inventor's own work practice, the pH value of the repair solution was set to 8.0. For the concentration value of the antibody and the optimal incubation time.

[0120] Considering that the antibodies used in the experiment after antibody comparison and selection are all from the same manufacturer and the signal strengths are the same, the two different antibodies are first mixed in a volume ratio of 1:1 for the experiment.

[0121] Then, according to the optimal incubation time of the primary antibody of 30 min and the optimal incubation time of the secondary antibody of 20 min in the above-mentioned double-antibody sequential staining, the staining is carried out.

[0122] Regarding the color development sequence and color development time of the color developer, combining the above-mentioned double-antibody sequential staining results, pre-experiment results and work experience, it is concluded that the sensitivity and stability of DAB in the two color developers are higher than those of RED. Therefore, when developing the color, RED is developed first for 10 min, and then DAB is developed for 5 min as the initial screening time.

[0123] Regarding the duration of hematoxylin counterstaining, considering that too long staining time of hematoxylin will cover the staining on the slide, the staining duration of hematoxylin in traditional immunohistochemistry is 10 min. Since the slide has been overlaid with two colors, the staining duration of hematoxylin needs to be shortened accordingly, but the situation of non-coloring should be avoided.

[0124] Based on this, a series of comparative experiments were designed in this embodiment, and some typical experimental groups and results are as follows in the table.

[0125] Table 3. Typical comparative experiments

[0126]

[0127] Subsequently, dehydration, mounting and microscopic examination were carried out according to the method of Example 1. The pathological sections of experimental groups a - e are respectively as Figures 5-9 shown.

[0128] It can be seen from the microscopic effect evaluation that for the fusion staining, when selecting the above-mentioned two antibodies of AE1 / AE3 mouse / S100 rabbit and AE1 / AE3 mouse / CD31 rabbit and carrying out the experimental conditions of experimental group c in the above table, the best effect is obtained and it is easy for diagnosticians to interpret.

[0129] (3) Multi-antibody fusion staining (AE1 / AE3 mouse / S100 rabbit / CD31 rabbit):

[0130] Referring to the experimental conditions of the above-mentioned double-antibody fusion staining, the mixed working solution of one of the primary antibodies is replaced with a mixed working solution containing 3 antibodies (AE1 / AE3 mouse / S100 rabbit / CD31 rabbit), specifically as follows:

[0131] Add the primary antibody mixed working solution (the volume ratio of AE1 / AE3, S-100, and CD31 is 1:1:1), incubate for a period of time (30 min) at an appropriate temperature (such as 37 °C), after incubation, rinse 2 - 3 times with the cleaning solution (30 s - 1 min each time), then add and add the corresponding secondary antibody mixed working solution (the volume ratio of mouse anti- and rabbit anti- is 1:1), incubate for a period of time (such as 20 min) at an appropriate temperature (such as 37 °C), after incubation, rinse 2 - 3 times with the cleaning solution (30 s - 1 min each time), then add the RED chromogenic solution and develop color for 10 min, add the DAB chromogenic solution and develop color for 10 min.

[0132] Subsequently, hematoxylin counterstaining, dehydration, mounting, and microscopic examination were carried out according to the method of Example 1. The obtained sections were as Figure 10 shown, where A is the area of nerve invasion in the section, and B is the area of blood vessel invasion in the section. It can be seen from the figure that AE1 / AE3 shows the epithelial-derived malignant tumor component as brown in the tissue section; S100 shows the nerve as a red solid structure in the tissue section, and CD31 shows the blood vessel as a red tubular structure in the tissue section. Therefore, even if the invaded tissues are all stained red, the malignant tumor component and the invaded tissue component can be clearly identified.

[0133] 2. Results

[0134] The above different experimental procedures were evaluated, and the results are shown in the following table.

[0135] Table 4. Evaluation and comparison table of different experimental procedures

[0136]

[0137] "Antibody selection limitation" means that the selection of two antibodies requires comparison, and only antibodies that do not affect each other can be used in combination.

[0138] Space resource savings = (((1) - (3)) The number of occupied experimental instrument space positions saved in each group / The number of occupied experimental instrument space positions by the conventional immunohistochemistry method) × 100%.

[0139] The conventional immunohistochemistry method is a method of performing AE1 / AE3 immunohistochemical staining, S-100 immunohistochemical staining, and CD31 immunohistochemical staining on tissue sections separately to obtain three sections.

[0140] One-time experiment success rate = (The number of successfully prepared slides in one time / The total number of samples) × 100%.

[0141] The number of paraffin block tissues consumed = The number of tissue sections that need to be cut for each method.

[0142] Effective interpretation rate = (Number of specimens with nerve invasion and vascular invasion that can be effectively interpreted by diagnosticians / (Number of samples * 2)) × 100%.

[0143] Cost savings are calculated as follows: ((Cost of preparing slides by the conventional immunohistochemistry method - Cost of preparing slides by the methods in each of groups (1) to (3)) / Cost of preparing slides by the conventional immunohistochemistry method) × 100%.

[0144] Improvement in process efficiency is calculated as follows: ((Duration of slide preparation and reading by the conventional immunohistochemistry method - Duration of slide preparation and reading by the methods in each of groups (1) to (3)) / Duration of slide preparation and reading by the conventional immunohistochemistry method) × 100%

[0145] The results showed that:

[0146] The effective interpretation rates and the success rates of a single experiment in the three groups of experiments (1), (2), and (3) were close, and the experimental effects were comparable.

[0147] Although there are limitations in antibody selection in groups (2) and (3) of the experiment, this problem can be solved by selecting the screened antibodies during fusion staining.

[0148] The preparation processes for the combined experiments of AE1 / AE3, S100, and CD31 in groups (1) and (2) of the experiment were more cumbersome and complex than those in group (3), consumed more tissue wax blocks, were not friendly to samples with relatively small lesion areas to be observed, and saved relatively less material cost and time cost.

[0149] Group (3) of the experiment can save material costs and space resources to a greater extent while ensuring a relatively high effective interpretation rate and a success rate of a single experiment, and greatly improve the efficiency of the experimental process, with the overall preparation process being simplified.

[0150] Therefore, the multi-antibody fusion staining (AE1 / AE3 rabbit / S100 mouse / CD31 mouse) in group (3) of the experiment was selected as the optimal staining scheme.

[0151] Example 3

[0152] In this example, the optimized multi-antibody fusion staining (AE1 / AE3 rabbit / S100 mouse / CD31 mouse) method obtained in Example 2 was further verified by scale-up.

[0153] 1. Method

[0154] According to the "multi-antibody fusion staining" method of the third group in Example 2, tissue sections were prepared from 85 samples of radical resection specimens of epithelial-derived malignant tumors, and AE1 / AE3 mouse antibody was overlaid with S-100 rabbit antibody and CD31 rabbit antibody, and the expression effects on epithelium, nerves, and blood vessels were observed and interpreted respectively.

[0155] Meanwhile, according to the conventional method, the slices were separately stained with AE1 / AE3, S-100, and CD31, and the observations and interpretations were carried out respectively.

[0156] 1.1 Verification of multi-antibody fusion staining (AE1 / AE3 rabbit / S100 mouse / CD31 mouse).

[0157] 1) Pretreatment before staining

[0158] ① Tissue sections: Take the tissue wax block and cut it into sections with a thickness of 2-3 μm.

[0159] ② Baking the sections: Bake the sections at 37-56 °C for 20-30 min.

[0160] ③ Dewaxing: Set the program on the machine, add 100 μl of dewaxing solution, temperature 72 °C, time 30 s.

[0161] Antigen retrieval: Set the program on the machine, add 100 μl of retrieval solution, temperature 100 °C, time 20 min. After retrieval, add peroxidase blocking agent for 12 min.

[0162] 2) Triple-antibody fusion staining

[0163] ① Add the primary antibody mixed working solution (volume ratio of mouse anti-AE1 / AE3: rabbit anti-S-100: rabbit anti-CD31 is 1:1:1), incubate at 37 °C for 30 min.

[0164] ② Add the secondary antibody: The secondary antibody is a mixed working solution of mouse anti- and rabbit anti-. Incubate at 37 °C for 20 min, and rinse with the washing solution 3 times, 3 min each time.

[0165] ③ Color development: Add RED and DAB color development solutions successively, develop color for 10 min each, and rinse with the washing solution 3 times, 3 min each time.

[0166] Hematoxylin counterstaining: Counterstain with hematoxylin for 5 min, and rinse with deionized water 3 times, 3 min each time.

[0167] Dehydration: Dehydrate successively with 80% ethanol for 3 min, 95% ethanol for 3 min, absolute ethanol for 3 min, and absolute ethanol for 3 min, and then place in 3 cylinders of TO for 3 min each for clearing treatment.

[0168] Mount with neutral balsam.

[0169] Examine the slides.

[0170] 1.2 Separate immunohistochemical AE1 / AE3 staining

[0171] The operation method is the same as above. When only adding the primary antibody mixed working solution, the added one is mouse anti-AE1 / AE3.

[0172] 1.3 Immunohistochemistry for S100 staining alone

[0173] The operation method is the same as above. When only adding the primary antibody mixed working solution, the added one is rabbit anti-S-100.

[0174] 1.4 Immunohistochemistry for CD31 staining alone

[0175] The operation method is the same as above. When only adding the primary antibody mixed working solution, the added one is rabbit anti-CD31.

[0176] For the separately stained slides, after the slide preparation is completed, the diagnostic physician views the original HE slide and the AE1 / AE3, S-100, and CD31 slides respectively, and repeatedly compares them to judge whether there is nerve and blood vessel invasion in the cancer tissue.

[0177] 2. Results

[0178] 2.1 Staining effect of the sections

[0179] Representative sections of multi-antibody fusion staining are as Figure 11 shown, which are photos of sections stained with multi-antibody fusion of rabbit anti-AE1 / AE3 / mouse anti-S100 / mouse anti-CD31.

[0180] Representative sections of single staining are as Figures 12-14 which are respectively photos of sections stained with AE1 / AE3, S-100, and CD31 alone.

[0181] From the above results, it can be seen that for the immunohistochemical multi-antibody fusion staining sections prepared by the present invention, the positive expression sites are accurate, the background is clear, the boundaries between the two colors at the positive sites are distinct, and there is no mutual interference, and the staining effect is not decreased compared with single staining.

[0182] 2.2 Judgment

[0183] Subsequently, the double-stained sections are judged by the pathologist according to the following table.

[0184] Table 5. Judgment of multi-antibody fusion staining for nerve and blood vessel invasion in malignant tumors of epithelial origin

[0185]

[0186] Taking the diagnostic results obtained by multiple antibody fusion staining of AE1 / AE3 rabbit / S100 mouse / CD31 mouse and single staining of AE1 / AE3, S-100, CD31 combined with HE by multiple diagnosticians as the standard results as a reference, 85 samples of radical resection specimens of poorly differentiated epithelial-derived malignant tumors were used, among which 21 cases were confirmed to have nerve invasion and 27 cases had vascular invasion. Calculate the reading accuracy rate.

[0187] Specifically, the observation and reading process for nerve and vascular invasion is as follows:

[0188] (I) Reading of single HE-stained sections

[0189] Nerve: The pathologist independently examined the HE-stained sections and preliminarily screened out that 56 cases were considered to have no nerve invasion, 13 cases were considered to have visible nerve invasion, and 16 cases could not be identified as having nerve invasion or not.

[0190] Vessel: The pathologist independently examined the HE-stained sections and preliminarily screened out that 33 cases were considered to have no vascular invasion, 25 cases were considered to have visible vascular invasion, and 27 cases could not be identified as having vascular invasion or not.

[0191] (II) Reading of the presence or absence of nerve and vascular invasion in epithelial-derived malignant tumors by single staining method

[0192] Nerve: The pathologist examined the HE, AE1 / AE3, and S-100 sections of the same sample. Among the 85 samples, in the 56 samples preliminarily screened and considered to have no nerve invasion, 54 cases had no nerve invasion and 2 cases had visible nerve invasion; in the 13 samples preliminarily screened and considered to have visible nerve invasion, 3 cases had no nerve invasion and 10 cases had visible nerve invasion; in the 16 samples preliminarily screened and unable to identify whether there was nerve invasion, 1 case could not be judged, 11 cases had no nerve invasion, and 4 cases had visible nerve invasion.

[0193] Vessel: The pathologist examined the HE, AE1 / AE3, and CD31 sections of the same sample. Among the 85 samples, in the 33 samples preliminarily screened (HE staining) and considered to have no vascular invasion, 30 cases had no vascular invasion and 3 cases had visible vascular invasion; in the 25 samples preliminarily screened and considered to have visible vascular invasion, 2 cases could not be judged, 13 cases had no vascular invasion, and 10 cases had visible vascular invasion; in the 27 samples preliminarily screened and unable to identify whether there was vascular invasion, 1 case could not be judged, 19 cases had no vascular invasion, and 7 cases had visible vascular invasion.

[0194] (III) Reading of the presence or absence of nerve and vascular invasion in epithelial-derived malignant tumors by triple antibody fusion staining method

[0195] The pathologist examined the HE, AE1 / AE3 / S-100 / CD31 sections of the same sample.

[0196] Nerves: For 85 samples, among the 56 samples where no nerve invasion was considered in the initial screening, 52 samples showed no nerve invasion and 4 samples showed nerve invasion. Among the 13 samples where nerve invasion was considered in the initial screening, 2 samples showed no nerve invasion and 11 samples showed nerve invasion. Among the 16 samples where it was impossible to identify whether there was nerve invasion in the initial screening, 10 samples showed no nerve invasion and 6 samples showed nerve invasion.

[0197] Vessels: For 85 samples, among the 33 samples where no vessel invasion was considered in the initial screening, 28 samples showed no vessel invasion and 5 samples showed vessel invasion. Among the 25 samples where vessel invasion was considered in the initial screening, 1 sample could not be interpreted, 12 samples showed no vessel invasion and 12 samples showed vessel invasion. Among the 27 samples where it was impossible to identify whether there was vessel invasion in the initial screening, 18 samples showed no vessel invasion and 9 samples showed vessel invasion.

[0198] Summarizing the above, the diagnostic physicians used different forms of sections for the evaluation and comparison of nerves and vessels. The statistical data is shown in Table 6 - 7 below, and the results are shown in Table 8 below.

[0199] Table 6 Statistical count of the number of cases for nerve invasion assessment

[0200]

[0201] Note: + indicates positive, showing invasion; - indicates negative, showing no invasion.

[0202] The above statistical results show that in the interpretation results of the separate HE staining method, the number of correctly interpreted cases was 63 (52 + 11) cases, and the number of cases that could not be interpreted was 16 cases.

[0203] In the interpretation results of the separate staining method, the number of correctly interpreted cases was 80 (52 + 2 + 2 + 10 + 10 + 4) cases, and the number of cases that could not be interpreted was 1 case.

[0204] In the interpretation results of the triple - antibody fusion staining method, the number of correctly interpreted cases was 85 (52 + 4 + 2 + 11 + 10 + 6) cases, and the number of cases that could not be interpreted was 0 cases.

[0205] Table 7 Statistical count of the number of cases for vessel invasion assessment

[0206]

[0207] Note: + indicates positive, showing invasion; - indicates negative, showing no invasion.

[0208] The above statistical results show that in the interpretation results of the separate HE staining method, the number of correctly interpreted cases was 41 (28 + 13) cases, and the number of cases that could not be interpreted was 27 cases.

[0209] The number of correctly interpreted cases in the results of the single staining method was 76 (28 + 3 + 11 + 10 + 17 + 7) cases, and the number of cases that could not be interpreted was 3 (2 + 1) cases.

[0210] The number of correctly interpreted cases in the results of the triple antibody fusion staining method was 83 (28 + 5 + 11 + 12 + 18 + 9) cases, and the number of cases that could not be interpreted was 1 case.

[0211] Table 8 Evaluation of nerve and blood vessel invasion in different forms of sections

[0212]

[0213] Note: Cases that cannot be interpreted are not classified as correct judgments.

[0214] Total accuracy rate = ((the number of cases with correct interpretation of nerve invasion + the number of cases with correct interpretation of blood vessel invasion) / (the number of cases × 2)) × 100%,

[0215] Total inefficiency rate = (the number of cases with nerve invasion that cannot be interpreted + the number of cases with blood vessel invasion that cannot be interpreted) / (the number of cases × 2)) × 100%,

[0216] The timeliness refers to the total time consumed for interpreting the sections for evaluating nerves and blood vessels of a single case.

[0217] The interpretation results showed that pathologists could accurately interpret both nerve and blood vessel invasions of malignant tumors of epithelial origin when interpreting samples of malignant tumors of epithelial origin prepared by using the multi - antibody fusion staining AE1 / AE3 rabbit / S100 mouse / CD31 mouse method, with an accuracy rate of 98.8%. However, when using only HE staining or comparing multiple HE, AE1 / AE3, S100, and CD31 sections, there were often cases that could not be interpreted, and the time consumed for interpretation was longer, with low diagnostic accuracy.

[0218] The results indicated that by using different chromogenic reagents to distinguish different antibodies on the same glass slide, the accuracy rate of evaluating the presence or absence of nerve and blood vessel invasions in samples of malignant tumors of epithelial origin could be improved, and diagnosticians could complete film reading more efficiently, greatly improving the efficiency and accuracy of diagnostic film reading.

Claims

1. An immunohistochemical multiple staining section, characterized in that: The multiple staining sections have epithelial malignant tumor staining marker antibodies and invaded tissue staining marker antibodies, the epithelial malignant tumor staining marker is AE1 / AE3 antibody, and the invaded tissue staining marker is one or more of S-100 antibody, CD31 antibody, CD34 antibody or NSE antibody.

2. The immunohistochemical multiple staining section according to claim 1, characterized in that: The invaded tissues are nerves and blood vessels, the staining and marking antibody for the nerves is S-100 antibody, and the staining and marking antibody for the blood vessels is CD31 antibody.

3. The immunohistochemical multiple staining section according to claim 1, characterized in that: The AE1 / AE3 antibodies are mouse antibodies, and the S-100 antibodies and CD31 antibodies are rabbit antibodies.

4. The method for preparing immunohistochemical multiple staining sections according to any one of claims 1 to 3, characterized in that: The following multiple staining steps are included: Take a tissue section, drop the epithelial malignant tumor staining antibody and the invaded tissue staining antibody, incubate with the primary antibody, then drop the secondary antibody that binds to the above antibodies, incubate with the secondary antibody, add the color developer for the above secondary antibody, color, and then perform hematoxylin counterstaining.

5. The method for preparing immunohistochemical multiple staining sections according to claim 4, characterized in that: Meet one or more of the following conditions: (1) In the multiple staining step, the ratio of the epithelial malignant tumor staining antibody to the invaded tissue staining antibody is 1:1; (2) In the multiple staining step, the epithelial malignant tumor staining antibody and the invaded tissue staining antibody are prepared as a mixed primary antibody solution for use; the mixed primary antibody solution is a mixed working solution of AE1 / AE3 antibody, S-100 antibody and CD31 antibody in a dosage ratio of 1:1:1; (3) In the multiple staining step, the secondary antibody is prepared as a mixed secondary antibody solution; (4) In the multiple staining step, the color developing agents are DAB and RED color developing solutions, and each color developing solution is used for 10 minutes respectively; (5) In the multiple staining step, the color developing agents are DAB and RED color developing solutions, and RED color development is performed first for 10 minutes, and then DAB color development is performed for 10 minutes; (6) In the multiple staining step, the hematoxylin counterstaining time is 5 minutes.

6. The method for preparing immunohistochemical multiple staining sections according to claim 4, characterized in that: Meet one or more of the following conditions: (1) In the multiple staining step, the primary antibody incubation temperature is 27°C-45°C; (2) In the multiple staining step, the primary antibody incubation time is 30 min; (3) In the multiple staining step, the temperature of the secondary antibody incubation is 27°C-45°C; (4) In the multiple staining step, the secondary antibody incubation time is 20 minutes.

7. The method for preparing immunohistochemical multiple staining sections according to claim 4, characterized in that: Before the multiple staining steps, a staining pre-treatment step is also included, and the staining pre-treatment step is as follows: taking the tissue to be stained, embedding and slicing, baking the slices, and then dewaxing the tissue slices and blocking the antigen repair peroxidase.

8. The method for preparing immunohistochemical multiple staining sections according to claim 4, characterized in that: After the multiple staining steps, a post-staining processing step is also included. The post-staining processing step is as follows: the stained sections are dehydrated and then sealed.

9. A method for pathological interpretation of epithelial malignant tumors for non-diagnostic treatment, characterized in that: The immunohistochemical multiple staining sections according to any one of claims 1 to 3 are used for interpretation: When the staining antibody for the invaded tissue is S-100 antibody or NSE antibody, the immunohistochemical multiple staining section is used to determine the invasion of the nerve by the epithelial malignant tumor; When the staining antibody for the invaded tissue is CD31 antibody or CD34 antibody, the immunohistochemical multiple staining sections are used to determine the invasion of blood vessels by epithelial malignant tumors.

10. A kit for pathological interpretation of epithelial malignant tumors, characterized in that: The method comprises the epithelial-derived malignant tumor staining antibody as described in any one of claims 1 to 3, the invaded tissue staining antibody as described in any one of claims 1 to 3, and the epithelial-derived malignant tumor staining antibody secondary antibody, the invaded tissue staining antibody secondary antibody, a colorimetric agent for the epithelial-derived malignant tumor staining antibody secondary antibody, and a colorimetric agent for the invaded tissue staining antibody secondary antibody; wherein the colorimetric agent for the epithelial-derived malignant tumor staining antibody secondary antibody is a DAB colorimetric agent, and the colorimetric agent for the invaded tissue staining antibody secondary antibody is a RED colorimetric agent.

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