Immunohistochemical multicolor display method under bright field microscope

Abstract

The invention belongs to the technical field of immunoassay, and particularly relates to an immunohistochemical multicolor display method under a bright field microscope. The method comprises the following steps: developing a to-be-detected tissue sample by adopting a color combination of yellow, red, green or yellow, purple and cyan blue in sequence; the to-be-detected tissue sample comprises a tumor lymph tertiary structure, infiltrating lymphocytes, urothelium cancer cells, non-small cell lung cancer and prostatic cancer. According to the color developing method of the color combination mode, qualitative and quantitative detection of the to-be-detected tissue can be achieved. According to the staining and developing method provided by the invention, the diagnosis time is saved, the diagnosis efficiency is improved, and signals which are originally required to be displayed on three pathological sections can be concentrated on one section to be displayed at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunoassay analysis, and in particular, relates to the application of a multicolor immunohistochemical display method under a bright field microscope in tumor diagnosis and immune microenvironment. Background Art

[0002] Bright field light microscope color development technology is different from the multi-color signal display under dark field fluorescence microscope. Its principle is to form a multi-color display by sequentially superimposing the original signals and finally synthesizing them by software. At present, only two-color (red and yellow) double labeling or two-color (red and yellow) triple labeling can be achieved in situ under bright field light microscope. This labeling technology has been reported in many aspects such as prostate cancer P63 / P504S or P63 / 34βE12 / P504S. However, its application is limited. At present, it can only provide one or two different colors of immunohistochemical display signals, which are relatively single and cannot make a comprehensive pathological diagnosis. The main reason is that it is impossible to display more tumor and environmental biomarkers. In addition, there are no literature reports on the light microscope display technology of immunohistochemistry with three-color or multi-color signals. One of the reasons may be that there are indeed technical difficulties, and the bottleneck of signal changes caused by the "subtraction principle" cannot be broken through. Summary of the invention

[0003] The primary purpose of the present invention is to provide a method for multicolor display of immunohistochemistry under a bright field microscope, so as to achieve the purpose of in situ multicolor display of tumors by naked eye observation through an optical microscope without the need for software synthesis.

[0004] To this end, the present invention provides the following technical solutions.

[0005] One aspect of the present invention provides a multicolor display method of immunohistochemistry under a bright field microscope, comprising the steps of developing the tissue samples to be tested using yellow, red, green, or a combination of yellow, purple, and cyan in sequence; the tissue samples to be tested include tumor lymphoid tertiary structures, infiltrating lymphocytes, urothelial cancer cells, non-small cell lung cancer, and prostate cancer.

[0006] As a preferred embodiment of the present invention, the method comprises the following steps: S1: Slice, dewax and wash the paraffin tissue to be tested to obtain tissue slices to be tested; S2: performing antigen repair on the tissue section to be tested to obtain the repaired tissue section to be tested; S3: The repaired tissue slice to be tested is stained using the following color combination method (1) or (2) to obtain a stained tissue slice: (1) Yellow, red, green; (2) Yellow, purple, and blue; S4: performing back staining on the obtained stained tissue sections, and after the back staining, dehydrating and sealing the tissue sections to obtain back staining tissue sections; S5: Observe the stained tissue sections under a microscope to obtain the color development results.

[0007] As a preferred embodiment of the present invention, in step S2, the antigen retrieval adopts EDTA-Tris pH 9.0.

[0008] As a preferred embodiment of the present invention, in step S2, the antigen repair conditions are: boiling in a 97-100°C water bath for 20-30 minutes, or jetting at 121°C and 1.21Kpa for 2-2.5 minutes.

[0009] As a preferred embodiment of the present invention, in step S3, the dyeing and coloring step specifically includes: (i) incubating the repaired tissue section with a first antibody and a second antibody in sequence, and then developing the color with a yellow dye to obtain a yellow tissue section; (ii) incubating the obtained yellow tissue sections with a third antibody and a second antibody in sequence, and then developing with a red or purple dye, and after the color development, soaking the tissue sections with acid to obtain tissue sections that are simultaneously yellow and red, or yellow and purple; (iii) The obtained tissue sections showing yellow and red, or yellow and purple, are incubated with a fourth antibody and a fifth antibody in sequence, and then developed with a green or cyan dye. After the color development is completed, tissue sections showing yellow, red and green, or yellow, purple and cyan are obtained.

[0010] As a preferred embodiment of the present invention, the first antibody is selected from mouse anti-human monoclonal antibody Ki67 or other cell nucleus localization antibodies; The second antibody includes HPR-polyplex secondary antibody EnVision; The third antibody is selected from mouse anti-human monoclonal antibody CK or other cytoplasmic localization antibodies; The fourth antibody is selected from mouse anti-human monoclonal antibody PD-L1 or other cell membrane localization antibodies; The fifth antibody includes AP-polyplex secondary antibody or HPR-polyplex secondary antibody EnVision.

[0011] As a preferred embodiment of the present invention, in step (i), the yellow dye comprises DAB.

[0012] As a preferred embodiment of the present invention, in step (ii), the red dye comprises HRP-Magenta.

[0013] As a preferred embodiment of the present invention, in step (ii), the purple dye comprises a Purple source.

[0014] As a preferred embodiment of the present invention, in step (ii), the acid is selected from any one of sulfuric acid, hydrochloric acid or acetic acid.

[0015] As a preferred embodiment of the present invention, in step (iii), the green dye includes Staygreen and Vinagreen.

[0016] As a preferred embodiment of the present invention, in step (iii), the cyan blue dye comprises Teal cyan blue.

[0017] As a preferred embodiment of the present invention, in step (iii), after color development, the tissue sections are immersed in dimethyl sulfoxide.

[0018] As a preferred embodiment of the present invention, in step S4, the dyes used for the lining dyeing include hematoxylin and nuclear fast red.

[0019] By means of the above technical solution, the present invention has at least the following advantages: 1. The present invention performs yellow, red, green and blue staining, or yellow, purple, cyan and red staining in sequence on the tissue sample to be tested. The two color combination staining method provided is to add equal to or greater than 3 color signal displays on the basis of the original method of two colors. The four color combinations and specific staining order provided by the present invention are all observation color combinations that the human eye is more sensitive to. They are easy to be observed and captured by the human eye under bright field of view to determine the degree of color contrast, and there is no interference from visual color difference, which ensures that the results only need to be displayed in situ by the naked eye through an optical microscope without the need for software synthesis. In addition, according to the evaluation criteria of immunohistochemistry, the staining method of the present invention is not only easy to qualitatively evaluate, but also easy to "quantitatively" evaluate the signal content.

[0020] 2. In the immunohistochemical multicolor display technology of the present invention, the green immunohistochemical substrates used include but are not limited to alkaline phosphatase substrates (StayGreen) and horseradish peroxidase substrates (Vinagreen), which all generate green signals with clear histological positioning, and the green signals are eye-catching; the red immunohistochemical substrates used include but are not limited to HRP enzymes (Magenta red substrates) and AP alkaline phosphatase substrates (Fastred, Permanentred), which, under the action of the corresponding enzymes, generate a pleasant red-pink color, which is easily observed by the observer. The two signals used in the present invention are stable and not easily decomposed by organic solvents to cause signal damage. In general clinical practice, after dozens of tests and the interpretation of more than 20 pathology experts, the color signal is generally reported to be good-looking (better than simple DAB yellow to brownish yellow), and the results are adopted.

[0021] 3. The present invention also overcomes the defect of attenuation or change of the original signal caused by the "subtraction principle" produced by two or more colors by using acid (including but not limited to 0.3M sulfuric acid) as the differentiation solution of HRP-Magenta red signal; using DMSO (dimethyl sulfoxide) as the differentiation solution of StayGreen green signal, thereby ensuring the objectivity and correctness of the signal distribution in the same field of view on a slice.

[0022] 4. The immunohistochemical multicolor display technology of the present invention is applicable to specimens of human or animal origin, including but not limited to formalin-fixed pathological masses, inflammatory tissues, cell smears, pleural effusions, etc. It is suitable for all clinical immunohistochemical staining automatic platforms. For example, the method of the present invention can realize the interpretation of the tumor microenvironment, including tumor lymphoid tertiary structure (TLS), infiltrating lymphocytes (TILs); it can also realize accurate companion diagnosis, including but not limited to PD-L1 CD23 CD8, etc.

[0023] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a micrograph of a human multi-organ tissue sample developed using the immunohistochemical multi-color display method of the present invention in Example 1; Figure 2 This is a micrograph of human multi-organ tissue samples developed with DAB, Magenta and StayGreen in sequence in Example 2, wherein brown-yellow indicates CKpan, red indicates Desmin, and green indicates CK7; Figure 3This is a micrograph of a human esophageal gland tissue sample developed with DAB, Magenta and StayGreen in turn in Example 3, wherein brown-yellow indicates Ki67, red indicates Desmin, and green indicates CKpan; Figure 4 This is a micrograph of a pig skin tissue sample developed with DAB, Magenta and StayGreen in turn in Example 4, wherein the brown-yellow color indicates Ki67, the red color indicates Desmin, and the green color indicates CKpan; Figure 5 This is a micrograph of the human immune microenvironment tissue sample using DAB, Purple source and Teal cyan blue in sequence in Example 5, wherein yellow is displayed as FOXP3, purple is displayed as CD20, and cyan blue is displayed as CD3; Figure 6 This is a micrograph of a pan-tumor lymphoid tissue sample developed with DAB, Magenta, and VinaGreen in sequence in Example 6, wherein yellow indicates PD-L1, green indicates CD23, and red indicates CD8; Figure 7 This is a micrograph of a human multi-organ tissue sample developed using a single DAB in Comparative Example 1; Figure 8 This is a micrograph of a human skin tissue sample developed using a single Magenta in Comparative Example 2; Fig. 9 This is a micrograph of a human prostate cancer tissue sample developed by DAB and Magenta in Comparative Example 3, wherein red is P504S and brown is P63; Fig.10 This is a micrograph of a human appendix tissue sample developed with DAB and Magenta in sequence in Comparative Example 4, wherein the brown-yellow color represents CKpan and the red color represents CD20. DETAILED DESCRIPTION

[0025] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] Example 1: Multicolor display of immunohistochemistry of human multi-organ tissue samples under bright field microscope 1. Cut the human multi-organ paraffin tissue into 2~3um slices, then dewax and finally wash them with water to obtain the human multi-organ tissue slices to be tested.

[0029] 2. Antigen repair procedure: EDTA-Tris pH 9.0 is used to perform antigen repair on the obtained human multi-organ tissue slices to be tested. The specific repair method is: the obtained human multi-organ tissue slices to be tested are placed in EDTA-Tris pH 9.0, heated in a water bath to boiling at 100°C and maintained for 20 minutes (according to common knowledge in the art, antigen repair can be carried out by a variety of methods well known in the art, including but not limited to vacuum negative pressure antigen repair method, high-pressure antigen repair method (121°C, 1.21Kpa pressure cooker with air jet for 2~2.5 minutes), etc. Only one method is described here, and other methods can also achieve the same purpose, which will not be repeated here). After the repair is completed, the tissue slices are cooled in water and finally rinsed with running water to obtain repaired tissue slices.

[0030] 3. Initial color development: After the above-mentioned repaired tissue sections were rinsed with PBS, the first antibody mouse anti-human monoclonal antibody Ki67 No. MIB1 (for immunohistochemistry) was added, and incubated in a 37°C incubator for 1 hour. After incubation, it was rinsed with PBS. Subsequently, the HPR-polyplex secondary antibody EnVision (DAKO No. K8001) was added, and incubated in a 37°C incubator for 20 minutes. After incubation, it was rinsed with PBS. Finally, the tissue sections were added to DAB (yellow) (1:50, prepared immediately before use, DAKO) for color development for 5 minutes to obtain yellow tissue sections.

[0031] 4. Secondary color development: After the tissue sections were washed with PBS after the initial color development, the second antibody mouse anti-human monoclonal antibody CK No. AE1 / AE3 (for immunohistochemistry) was added, and incubated in a 37°C incubator for 1 hour. After incubation, it was rinsed with PBS, and then HRP-polyplex secondary antibody EnVision (DAKO No. K8001) was added. It was incubated in a 37°C incubator for 20 minutes. After incubation, it was rinsed with PBS, and the tissue sections were added to HRP-Magenta (red) (17:3, prepared immediately before use, DAKO) for color development for 5 minutes. Finally, the tissue sections were soaked in 0.3M sulfuric acid for 3 minutes to obtain tissue sections that were simultaneously yellow and red.

[0032] 5. Tertiary color development: After the tissue sections after the secondary color development were rinsed with PBS, the third antibody, mouse anti-human monoclonal antibody Desmin, No. D33 (for immunohistochemistry) was added, and incubated in a 37°C incubator for 1 hour. After incubation, it was rinsed with PBS. Subsequently, AP-polyplex secondary antibody (Roche No. 760-501) was added, and incubated in a 37°C incubator for 20 minutes. After incubation, it was rinsed with PBS, and the tissue sections were added to Staygreen (green) (1:50, prepared immediately before use, abcam) for color development for 15 minutes. Finally, the tissue sections were soaked in DMSO (dimethyl sulfoxide, Sigma) for 3 minutes to obtain tissue sections that were simultaneously yellow, red and green.

[0033] 6. Staining and sealing: After the obtained yellow, red and green tissue sections are rinsed with running water, they are stained with hematoxylin for 30 seconds, then dehydrated with titrated alcohol, and finally sealed to obtain stained tissue sections.

[0034] 7. Microscope observation: Place the stained tissue sections under a microscope for observation, and confirm whether there is signal expression based on the color of the different substrates / products. Figure 1 .

[0035] like Figure 1 As shown, by sequentially using the yellow, red, and green three-color development techniques on human multi-organ tissue slices, a tissue slice staining image in which red, green, and yellow signals are displayed simultaneously can be obtained, and each color is distinct and does not interfere with each other, making it easy for the operator to interpret.

[0036] Example 2: Multicolor display of human multi-organ tissue samples by immunohistochemistry under bright field microscope The difference from Example 1 is that StayGreen, DAB and Magenta are used in sequence to perform primary, secondary and tertiary color development on human multi-organ tissue sample slices, and the rest is the same as Example 1.

[0037] Microscopic observation results are shown in Figure 2 .like Figure 2 As shown, liver hepatocytes and kidney ducts show yellow to brownish-yellow signals of CKpan, ovarian and appendiceal muscles show rose-red signals of Desmin, and amniotic membrane, liver bile duct, kidney distal convoluted tubules and prostate epithelium show green to dark green signals of CK7.

[0038] Example 3: Multicolor Immunohistochemical Display of Human Esophageal Gland Tissue Samples under Bright Field Microscopy The difference from Example 1 is that DAB, Magenta and StayGreen are used in sequence to perform primary, secondary and tertiary color development on the human esophageal gland tissue sample sections, and the rest is the same as Example 1.

[0039] Microscopic observation results are shown in Figure 3 .like Figure 3 As shown, scattered lymphocytes in the oral cavity present yellow to brownish-yellow Ki67 signals, oral muscle tissue presents rose-red Desmin signals, and secretory gland epithelium presents green to dark green CKpan signals.

[0040] Example 4: Multicolor Immunohistochemical Display of Pig Skin Tissue Samples under Bright Field Microscopy The difference from Example 1 is that DAB, Magenta and StayGreen are used in sequence to perform primary color development, secondary color development and tertiary color development on the pig skin tissue sample section, and the rest is the same as Example 1.

[0041] Microscopic observation results are shown in Figure 4 .like Figure 4 As shown, the basal cells of the skin and scattered lymphocytes in the muscular layer showed yellow to brownish-yellow Ki67 signals, the subcutaneous muscle tissue showed rose-red Desmin signals, and the subcutaneous secretory gland epithelium showed green to dark green CKpan signals.

[0042] Example 5: Multicolor display of immunohistochemistry of human immune microenvironment tissue samples under bright field microscope The difference from Example 1 is that DAB, Purple source and Teal blue are used in sequence to perform primary, secondary and tertiary color development on the human immune microenvironment tissue sample slices, and the rest is the same as Example 1.

[0043] Microscopic observation results are shown in Figure 5 .like Figure 5 As shown, the nucleus of regulatory T cells shows yellow FOXP3 signals, B lymphocytes show purple CD20 signals, and scattered T cells show cyan CD3 signals.

[0044] Example 6: Multicolor Immunohistochemical Display of Pan-tumor Lymphoid Tissue Samples under Bright Field Microscopy The difference from Example 1 is that DAB, Magenta, and VinaGreen are used in sequence to perform primary, secondary, and tertiary color development on pan-tumor (including but not limited to gastric cancer, intestinal cancer, lung cancer, esophageal cancer, and breast cancer) lymphoid tissue-like sections, and the rest is the same as Example 1.

[0045] Microscopic observation results are shown in Figure 6 .like Figure 6 As shown, epithelial cells and macrophages show PD-L1 yellow (DAB) signal, follicular dendritic network and B cells show CD23 green (VinaGreen) signal in the germinal center, and 1 TLS (tertiary lymphoid structure) is seen; T lymphocytes with CD8 red (Magenta) signal can be seen around the TLS.

[0046] Comparative Example 1: Using a single DAB to color multiple human organ tissues The difference from Example 1 is that only DAB is used for the initial color development of human multi-organ tissue slices, and the rest is the same as Example 1.

[0047] Microscopic observation results are shown in Figure 7 .like Figure 7 As shown, the epithelial cells such as kidney and placenta only show a single CK signal of yellow to brownish yellow.

[0048] Comparative Example 2: Using a single Magenta to develop color on human skin tissue samples The difference from Example 1 is that only Magenta is used to perform the initial color development on the human skin tissue slices, and the rest is the same as Example 1.

[0049] Microscopic observation results are shown in Figure 8 .like Figure 8 As shown, the skin epithelium only presents a single CK rose-red signal.

[0050] Comparative Example 3: Using DAB and Magenta to color human prostate cancer tissue samples The difference from Example 1 is that only DAB and Magenta are used to perform primary and secondary color development on human prostate cancer tissue sections, and the rest is the same as Example 1.

[0051] Microscopic observation results are shown in Fig. 9 .like Fig. 9 As shown, prostate myoepithelial cells present yellow to brownish yellow signals of P63, and prostate cancer epithelial cells present rose-red signals of P504S.

[0052] Comparative Example 4: Using DAB and Magenta to color human appendix tissue samples The difference from Example 1 is that only DAB and Magenta are used to perform primary and secondary color development on human appendix tissue sections, and the rest is the same as Example 1.

[0053] Microscopic observation results are shown in Fig.10 .like Fig.10 As shown, the appendix mucosal epithelial cells showed CK yellow to brownish yellow signals, and the lymphocytes in the lamina propria showed CD20 rose-red signals.

[0054] It can be seen from the above results that, compared with the monochrome signal display of Comparative Examples 1 and 2, and the dual-color signal display of Comparative Examples 3 and 4, the three-color signals and specific staining order of Examples 1-6 of the present invention have clearer and more distinct signal display. In addition, the signal display method of Examples 1-6 of the present invention can not only make qualitative judgments on the tissue to be tested, but also can realize quantitative detection of tissue cells, for example, the pathologist can count tumor cells and different types of surrounding immune cells with the naked eye, and describe the "good or bad" of the immune environment through digital ratios, and can display parameters such as tumor diameter and overall area.

[0055] Specific application: Clinical application of the immunohistochemical multicolor display method of the present invention Based on the construction of multi-color in bright field, the workload of the reader is reduced, and multiple results can be described more accurately. Compared with the existing two-color bright field display technology and dark field fluorescence multi-color display technology, the bright field multi-color signal display technology of the present invention is more conducive to clear diagnosis and tumor biological information observation.

[0056] By using the bright field multicolor color development technology, dual-color bright field display technology (commercial), and dark field fluorescence multicolor display technology (scientific research) of the present invention to stain and color NSCLC tumor tissue samples in clinical practice, the results show that compared with the other two color development technologies, the bright field multicolor color development technology of the present invention can achieve the interpretation of tumor microenvironment, accurate interpretation of companion diagnosis, and tumor diagnosis, etc. Specific comparison information is shown in Table 1: Table 1

[0057] Note: NSCLC refers to non-small cell lung cancer.

[0058] From the results in Table 1 above, it can be seen that the bright field multicolor display technology of the present invention can be used to observe tumor epithelial cell CK and microenvironment including the lymphatic tertiary structure TLS (mainly including B cell-CD20, follicular dendritic cells (FDC-CD23, T cell-CD4, CD8, macrophage-CD68 and PD-1 PD-L1 pathway). This allows pathologists to make accurate judgments and decisions in situations they are familiar with.

[0059] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with this profession can make some changes or modifications to equivalent embodiments of equivalent changes by using the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A multicolor display method of immunohistochemistry under a bright field microscope, characterized in that: The method comprises the steps of sequentially developing the color of the tissue sample to be tested using yellow, red, green, or a combination of yellow, purple, and cyan; The tissue samples to be tested include tumor lymphoid tertiary structures, infiltrating lymphocytes, urothelial cancer cells, non-small cell lung cancer, and prostate cancer.

2. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 1, characterized in that: The method comprises the following steps: S1: Slice, dewax and wash the paraffin tissue to be tested to obtain tissue slices to be tested; S2: performing antigen repair on the tissue section to be tested to obtain the repaired tissue section to be tested; S3: Stain the repaired tissue slice to be tested using the following color combination method (1) or (2) to obtain a stained tissue slice: (1) Yellow, red, green; (2) Yellow, purple, and blue; S4: performing back staining on the obtained stained tissue sections, and after the back staining, dehydrating and sealing the tissue sections to obtain back staining tissue sections; S5: Observe the stained tissue sections under a microscope to obtain the color development results.

3. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 2, characterized in that: In step S2, the antigen retrieval is performed using EDTA-Tris pH 9.

0.

4. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 2, characterized in that: In step S2, the antigen repair conditions are: boiling in a water bath at 97-100°C for 20-30 min, or jetting at 121°C and 1.21 KPa for 2-2.5 min.

5. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 2, characterized in that: In step S3, the dyeing and coloring step specifically includes: (i) incubating the repaired tissue section with a first antibody and a second antibody in sequence, and then developing the color with a yellow dye to obtain a yellow tissue section; (ii) incubating the obtained yellow tissue sections with a third antibody and a second antibody in sequence, and then developing with a red or purple dye, and after the color development, soaking the tissue sections with acid to obtain tissue sections that are simultaneously yellow and red, or yellow and purple; (iii) The obtained tissue sections showing yellow and red, or yellow and purple, are incubated with a fourth antibody and a fifth antibody in sequence, and then developed with a green or cyan dye. After the color development is completed, tissue sections showing yellow, red and green, or yellow, purple and cyan are obtained.

6. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 5, characterized in that: The first antibody is selected from mouse anti-human monoclonal antibody Ki67 or other cell nucleus localization antibodies; The second antibody includes HPR-polyplex secondary antibody EnVision; The third antibody is selected from mouse anti-human monoclonal antibody CK or other cytoplasmic localization antibodies; The fourth antibody is selected from mouse anti-human monoclonal antibody PD-L1 or other cell membrane localization antibodies; The fifth antibody includes AP-polyplex secondary antibody or HPR-polyplex secondary antibody EnVision.

7. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 5, characterized in that: In step (i), the yellow dye comprises DAB.

8. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 5, characterized in that: In step (ii), the red dye comprises HRP-Magenta; The violet dye includes a Purple source.

9. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 5, characterized in that: In step (iii), the green dye includes Staygreen and Vinagreen; The cyan blue dye includes Teal cyan blue.

10. The method for multicolor display of immunohistochemistry under bright field microscope according to claim 2, characterized in that: In step S4, the dyes used for the lining dyeing include hematoxylin and nuclear fast red.

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