Multiple fluorescent staining method for multiple protein targets

Abstract

The invention discloses a multiple fluorescent staining method for multiple protein targets, and relates to the technical field of multiple immunofluorescent staining. According to the method provided by the invention, the number of dyeable protein targets in the tissue slice can be greatly increased, multiple fluorescent staining steps can be simplified, and by adopting the method provided by the invention, imaging can be carried out after all the targets are directly stained on the basis of one tissue slice. The number of tissue slices required by dyeing imaging is reduced, dyeing of more than 6 protein targets can be completed by only one tissue slice, and errors caused by slice continuity differences are avoided. In addition, repeated elution and re-dyeing are not needed, so that the problems of antigen loss and weakening and the like caused by repeated elution are avoided.

Description

Technical Field

[0001] This invention relates to the field of multiplex immunofluorescence staining technology, and more specifically, to a multiplex fluorescence staining method for multiple protein targets. Background Technology

[0002] The tumor microenvironment (TME) is a crucial factor in tumor development, progression, response, and anti-tumor therapy, playing a significant role in tumor prognosis and the efficacy of anti-tumor drugs. The immune environment within the tumor microenvironment has also garnered considerable attention; for example, in anti-tumor drug development, drugs targeting immune checkpoints are frequently developed, and when successful, they typically produce durable anti-tumor effects. The emergence of tumor immunotherapy has revolutionized traditional cancer treatment, making the research and identification of biomarkers related to the efficacy of immune checkpoint inhibitors particularly urgent and important.

[0003] Multiplex immunohistochemistry (mIHC) can simultaneously assess the expression of multiple biomarkers in the same tissue section, serving as a powerful tool for tumor immunology and biomarker research. Multiplex immunohistochemistry primarily utilizes antibodies labeled with different markers to identify target proteins on tissue sections, and then uses different experimental platforms or specialized instruments to acquire and analyze images. Due to the wealth of biological information obtained, it can analyze not only the types, components, and expression levels of in situ targets in tissue cells, but also the spatial location information of interactions between various targets. mIHC technology plays an important role in studies of the tumor microenvironment, tumor heterogeneity, and tumor development and progression.

[0004] Currently, commercially available multicolor immunohistochemistry kits can only label six target sites on a single slide and perform full-slide imaging. If researchers want to explore the staining relationships of more target sites, the mainstream methods are sequential staining followed by overlay imaging and restaining after imaging and elution. Both methods have certain drawbacks. Sequential staining followed by overlay imaging suffers from errors due to differences in slide continuity, while restaining after imaging and elution can lead to antigen loss and weakening after repeated elution. Therefore, increasing the number of stainable target sites on a single slide in multiplex immunohistochemistry experiments has become a pressing issue in this field.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a multiplex fluorescence staining method for multiple protein targets to solve the above-mentioned technical problems.

[0007] This invention is implemented as follows:

[0008] This invention provides a multiplex fluorescence staining method for multiple protein targets, which includes the following steps: using multiple combinations of fluorophores to stain the sample to be tested sequentially, each combination of fluorophores including two fluorophores with different emission wavelengths, and at least two fluorophores in any two combinations of fluorophores having different emission wavelengths;

[0009] In the staining step, one fluorochrome combination corresponds to one protein target site, and there are at least 7 protein targets.

[0010] The method provided by this invention can significantly increase the number of stainable protein targets in tissue sections and simplify the multiplex fluorescence staining process. Existing techniques require imaging after section staining; for imaging of more than seven protein targets, it is necessary to first image the fluorescence of two or more of these targets, and then stain and image the remaining protein targets on another tissue section obtained from consecutive sections, creating a superimposed image. Using the method provided by this invention, imaging can be performed directly after staining all targets on a single tissue section. This saves on the number of tissue sections required for staining and imaging; more than six protein targets can be stained with just one tissue section, avoiding errors caused by differences in section continuity. Furthermore, it eliminates the need for repeated elution and re-staining, avoiding problems such as antigen loss or weakening due to repeated elution.

[0011] The number of protein targets is not limited to 7, but can range from 7 to 20, including but not limited to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0012] It should be noted that fluorescein refers to dyes that exhibit photofluorescence properties.

[0013] In a preferred embodiment of the present invention, the aforementioned fluorescein combination is selected from combinations of fluorescein with emission wavelengths in the range of 465-780 nm. Any fluorescein having the aforementioned emission wavelengths can constitute a component unit of the fluorescein combination.

[0014] In a preferred embodiment of the present invention, the above-mentioned fluorescein combination is selected from at least 7 fluorescein combinations formed by at least 5 fluorescein emission wavelengths selected from 465-700nm.

[0015] In a preferred embodiment of the present invention, the above-mentioned fluorophore combination is selected from at least seven fluorophore combinations formed by at least five fluorophores with emission wavelengths of 465nm (e.g., FAM), 480nm, 490nm (5-FAM), 515nm (5(6)-FAM), 520nm (e.g., FITC), 570nm, 620nm, 690nm, 700nm, and 780nm.

[0016] In a preferred embodiment of the present invention, the sample to be tested is stained sequentially using the following combination of fluorescein emission wavelengths:

[0017] The staining sequences are 480nm+520nm, 480nm+570nm, 480nm+620nm, 480nm+690nm, 520nm+570nm, 520nm+620nm, 520nm+690nm, 570nm+620nm, 570nm+690nm, and 620nm+690nm. With these staining sequences, 10 protein target sites can be stained in the same tissue section without color mixing or affecting imaging. In other embodiments, those skilled in the art can adjust the staining sequence as needed.

[0018] In a preferred embodiment of the invention, the fluorescein is selected from the Opal 6-Plex Detection Kit for Whole Slide Imaging manufactured by Akoya Biosciences. In other embodiments, the fluorescein may also be selected from fluorescein kits from other companies.

[0019] In a preferred embodiment of the present invention, the above-mentioned fluorescein combination is selected from at least five fluorescein combinations selected from FITC, PE, APC, Alexa Fluor 647, Alexa Fluor 555, Alexa Fluor 488, FAM, 5-FAM, 5(6)-FAM, HEX, JOE, Lissamine Rhodamine B, Pyrene, ROX, TET, Tetramethyl Rhodamine, Texas Red, Dabcyl, CR-6G, BODIPY R6G, BODIPY FL, Amino-coumarin, Opal 480Reagent, Opal 520Reagent, Opal 570Reagent, Opal 620Reagent, and Opal 690Reagent.

[0020] In a preferred embodiment of the present invention, the above-mentioned multiplex fluorescent staining is performed using the Leica Bond RX platform.

[0021] In a preferred embodiment of the present invention, the above-mentioned multiplex fluorescent staining method includes the following steps performed sequentially: antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, incubation with the first fluorescein in the fluorescein combination, and incubation with the second fluorescein in the same fluorescein combination; then, changing to another fluorescein combination, and performing antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, and fluorescein incubation in one step; until the target fluorescein is stained onto the sample to be stained;

[0022] In a preferred embodiment of the present invention, the sample to be stained is a tissue section or a clinical smear.

[0023] Tissue sections include, but are not limited to, sections of the brain, adrenal glands, colon, small intestine, stomach, heart, liver, skin, kidneys, lungs, pancreas, testes, ovaries, prostate, uterus, thyroid gland, and spleen of mammals. Frozen sections are especially important.

[0024] The tissue sections mentioned above are obtained through surgical excision or puncture of living tissue; or are selected from coarse needle biopsy tissue sections or small biopsy tissue sections.

[0025] In a preferred embodiment of the present invention, the antigen retrieval refers to: treating the sample with an antigen retrieval solution and treating the dewaxed sample to be stained at 95-97°C for 10-30 minutes.

[0026] During tissue preparation, the antigens are blocked by the reagents, and the peptide chains of some antigens are twisted due to the heat, making them unable to be displayed during immunohistochemical staining. To solve the above problems, the process of re-exposing or correcting these antigens using chemical reagents and heat is called antigen retrieval.

[0027] The incubation conditions for any one of the luciferin combinations are incubation at room temperature for 10 minutes. In a preferred embodiment of the present invention, incubation is performed at 6°C-28°C for 10 minutes.

[0028] To achieve better incubation results with primary antibodies, those skilled in the art can set the number of incubation cycles and the incubation time as needed. For example, for antibodies that are difficult to label with primary antibodies, multiple labeling cycles can be performed to ensure that the slide sample is labeled with primary antibodies. For example, incubation with primary antibodies can be performed 1-4 times.

[0029] The present invention has the following beneficial effects:

[0030] The method provided by this invention can significantly increase the number of stainable protein targets in tissue sections and simplify the steps of multiplex fluorescence staining. Existing technologies require imaging after section staining. For imaging of more than 7 protein targets, it is necessary to first image the fluorescence images of 2 or more of the protein targets, and then stain and image the other protein targets on another tissue section sample obtained from consecutive sections. The overlay process includes: acquiring staining images of different targets in consecutive sections → using the HALO Deconvolution module to convert the IHC image into a pseudo-fluorescent image (if the original image is a fluorescent image, the conversion step can be skipped) → manually adjusting the image magnification and angle. Figure 11 → The software performs secondary image correction → Parameter settings and image generation ( Figure 12 ));

[0031] The method provided by this invention allows for imaging after staining all target sites on a single tissue section. This reduces the number of tissue sections required for staining and imaging, as more than six protein target sites can be stained with just one tissue section, avoiding errors caused by differences in section continuity. Furthermore, it eliminates the need for repeated elution and re-staining, avoiding problems such as antigen loss or weakening due to repeated elution. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 Image showing the staining effect of CD4 (480nm+520nm) (magnification 200X);

[0034] Figure 2 This is a staining effect image of CD8 (480nm+570nm) (magnification 200X);

[0035] Figure 3 This is a PD-L1 (480nm+620nm) staining effect image (magnification 200X);

[0036] Figure 4 This is a staining effect image of CD80 (480nm+690nm) (magnification 200X);

[0037] Figure 5 This is a staining effect image of VIM (520nm+570nm) (magnification 200X);

[0038] Figure 6 This is an image showing the staining effect of Grzb (520nm+620nm) (magnification 200X);

[0039] Figure 7 This is an image showing the staining effect of CD206 (520nm+690nm) (magnification 200X);

[0040] Figure 8 This is a staining effect image of CD163 (570nm+620nm) (magnification 200X);

[0041] Figure 9 This is a staining effect image of B7-H3 (570nm+690nm) (magnification 200X);

[0042] Figure 10 This is a staining effect image of FOXP3 (620nm+690nm) (magnification 200X);

[0043] Figure 11 This is a flowchart of existing technology for image conversion of IHC images;

[0044] Figure 12 This is a flowchart of existing techniques for overlaying pseudo-fluorescent images. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0046] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0047] Example 1

[0048] In this embodiment, 10 protein targets were fluorescently stained using five fluorophores (480nm, 520nm, 570nm, 620nm, 690nm) from the Opal 6-Plex Detection Kit for WholeSlide Imaging manufactured by AkoyaBio.

[0049] The instruments, reagents, and main procedures involved are shown in Tables 1, 2, and 3.

[0050] The multiplex fluorescence staining method for multiple protein targets specifically includes the following steps:

[0051] 1. In this embodiment, the tissue section to be stained is a paraffin section from commercially available non-small cell lung cancer tissue.

[0052] 2. Place the tissue sections into the fully automated immunohistochemistry and in situ hybridization staining machine and execute the following procedure:

[0053] (1) Dewaxing: Dewaxing the stained sections using dewaxing solution at 72℃ for 30 minutes.

[0054] (2) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the dewaxed sections. The treatment conditions were 97℃ for 20 min.

[0055] (3) Blocking: PKI Blocking Buffer (Opal 6-Plex Detection Kit for WholeSlide Imaging component, catalog number: NEL871001KT) was used for blocking at room temperature for 10 min.

[0056] (4) Primary antibody incubation: Antibody incubation was performed using rabbit anti-CD4 antibody at room temperature for 30 min.

[0057] (5) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP (Opal 6-Plex Detection Kit for WholeSlide Imaging component, catalog number: NEL871001KT) at room temperature for 10 min.

[0058] (6) Fluorescein incubation: Fluorescein incubation was performed using Opal 480 Reagent at room temperature for 10 min;

[0059] (7) Fluorescein incubation: Fluorescein was incubated using Opal 520 Reagent at room temperature for 10 min;

[0060] (8) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0061] (9) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 minutes.

[0062] (10) Primary antibody incubation: Antibody incubation was performed using rabbit anti-CD8 antibody at room temperature for 30 min.

[0063] (11) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0064] (12) Fluorescein incubation: Fluorescein was incubated using Opal 480 Reagent at room temperature for 10 min;

[0065] (13) Fluorescein incubation: Fluorescein incubation was performed using Opal 570 Reagent at room temperature for 10 min;

[0066] (14) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0067] (15) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0068] (16) Primary antibody incubation: Antibody incubation was performed using rabbit anti-PD-L1 antibody at room temperature for 30 min.

[0069] (17) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0070] (18) Fluorescein incubation: Fluorescein incubation was performed using Opal 480 Reagent at room temperature for 10 min;

[0071] (19) Fluorescein incubation: Fluorescein was incubated using Opal 620 Reagent at room temperature for 10 min;

[0072] (20) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0073] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0074] (21) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0075] (22) Primary antibody incubation: Antibody incubation was performed using anti-CD-80 antibody at room temperature for 30 min.

[0076] (23) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0077] (24) Fluorescein incubation: Fluorescein was incubated using Opal 480 Reagent at room temperature for 10 min;

[0078] (25) Fluorescein incubation: Fluorescein incubation was performed using Opal 690 Reagent at room temperature for 10 min;

[0079] (26) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0080] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0081] (27) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0082] (28) Primary antibody incubation: Antibody incubation was performed using anti-VIM antibody at room temperature for 30 min.

[0083] (29) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0084] (30) Fluorescein incubation: Fluorescein was incubated using Opal 520 Reagent at room temperature for 10 min;

[0085] (31) Fluorescein incubation: Fluorescein incubation was performed using Opal 570 Reagent at room temperature for 10 min;

[0086] (32) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0087] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0088] (33) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0089] (34) Primary antibody incubation: Antibody incubation was performed using anti-Grzb antibody at room temperature for 30 min.

[0090] (35) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0091] (36) Fluorescein incubation: Fluorescein incubation was performed using Opal 520 Reagent at room temperature for 10 min;

[0092] (37) Fluorescein incubation: Fluorescein was incubated using Opal 620 Reagent at room temperature for 10 min;

[0093] (38) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0094] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0095] (39) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0096] (40) Primary antibody incubation: Antibody incubation was performed using anti-CD206 antibody at room temperature for 30 min.

[0097] (41) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0098] (42) Fluorescein incubation: Fluorescein was incubated using Opal 520 Reagent at room temperature for 10 min;

[0099] (43) Fluorescein incubation: Fluorescein incubation was performed using Opal 690 Reagent at room temperature for 10 min;

[0100] (44) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0101] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0102] (45) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0103] (46) Primary antibody incubation: Antibody incubation was performed using anti-CD163 antibody at room temperature for 30 min.

[0104] (47) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0105] (48) Fluorescein incubation: Fluorescein incubation was performed using Opal 570 Reagent at room temperature for 10 min;

[0106] (49) Fluorescein incubation: Fluorescein was incubated using Opal 620 Reagent at room temperature for 10 min;

[0107] (50) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0108] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0109] (51) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0110] (52) Primary antibody incubation: Antibody incubation was performed using anti-B7-H3 antibody at room temperature for 30 min.

[0111] (53) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0112] (54) Fluorescein incubation: Fluorescein incubation was performed using Opal 570 Reagent at room temperature for 10 min;

[0113] (55) Fluorescein incubation: Fluorescein incubation was performed using Opal 690 Reagent at room temperature for 10 min;

[0114] (56) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 10 min.

[0115] Antigen retrieval was performed again: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0116] (57) Blocking: PKI Blocking Buffer was used for blocking at room temperature for 10 min.

[0117] (58) Primary antibody incubation: Antibody incubation was performed using anti-FOXP3 antibody at room temperature for 60 min.

[0118] Repeat the primary antibody incubation process twice more, for a total of three primary antibody incubations.

[0119] (59) Secondary antibody incubation: Secondary antibody incubation was performed using Opal Polymer HRP at room temperature for 10 min.

[0120] (60) Fluorescein incubation: Fluorescein was incubated using Opal 620 Reagent at room temperature for 10 min;

[0121] (61) Fluorescein incubation: Fluorescein incubation was performed using Opal 690 Reagent at room temperature for 10 min;

[0122] (62) Antigen retrieval: Antigen retrieval solution 2 was used to retrieval the antigens on the above sections. The treatment conditions were 95℃ for 20 min.

[0123] (63) Counterstaining: The above sections were counterstained with Spectral DAPI reagent at room temperature for 10 min.

[0124] The software is used to set up the "reagent settings", "slide settings", and "program settings" to determine the staining procedure. Labels are printed, and the corresponding tissue sections are labeled. The labeled slides and reagents are then placed into the staining machine for quality inspection. After passing the quality inspection, the staining procedure described above is initiated.

[0125] Table 1 Instruments and Equipment

[0126] name factory model Fully automated immunohistochemistry and in situ hybridization staining machine Leica Bond RX Fully automatic staining and sealing machine Dakota DP360-CS500 Fluorescence imaging system Akoya

[0127] Table 2 Reagent Information

[0128]

[0129] Table 3 Staining Procedure

[0130]

[0131]

[0132]

[0133] The cleaning steps are not listed in Table 3. The fully automated immunohistochemistry and in situ hybridization staining machine has a cleaning step by default.

[0134] The staining effect images of 10 different target sites after staining 10 protein target sites in the above tissue sections are shown in the figure. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 and Figure 10 As shown in the figure. The results show that the 20-fold fluorescence staining for 10 different protein targets provided by this invention has excellent staining effect.

[0135] In summary, this embodiment utilizes the arrangement and combination of fluorophores in the Opal 6-Plex Detection Kit for Whole Slide Imaging manufactured by AkoyaBio, and uses two fluorophores to label the same target point. This enables the achievement of a staining capability on a single, fully scannable slide that is far greater than the 6-label staining capability of the Opal 6-Plex Detection Kit for Whole Slide Imaging kit, allowing for the observation of more target staining relationships within the same slide.

[0136] Result Comparison Statistics Table:

[0137] staining target Fluorescent staining percentage % Bright field staining percentage CV value CD4 5.22 5.22 5.18％ CD8 5.64 5.64 5.77％ PD-L1 71.63 71.63 5.30％ CD80 81.17 81.17 13.78％ VIM 5.64 4.52 9.84％ Grzb 81.17 77.34 1.98％ CD206 15.08 16.29 9.77％ CD163 1.54 1.99 15.65％ B7-H3 74.23 73.33 0.61％ FOXP3 7.54 7.28 1.79％

[0138] The inventors declare that this invention illustrates a method for staining six or more target points using the Opal 6-Plex Detection Kit for Whole Slide Imaging manufactured by AkoyaBio through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials in the product of this invention, additions of auxiliary components, and selection of specific methods all fall within the protection and disclosure scope of this invention.

Claims

1. A multiplex fluorescence staining method for multiple protein targets, characterized in that, It includes the following steps: sequentially staining the sample to be tested with multiple fluorophore combinations, each fluorophore combination including two fluorophores with different emission wavelengths, and at least two fluorophores in any two fluorophore combinations having different emission wavelengths; In the staining step, one fluorescein combination corresponds to one protein target site, and there are at least 7 protein targets.

2. The multiplex fluorescent staining method according to claim 1, characterized in that, The fluorochrome combination is selected from fluorochrome combinations with emission wavelengths of 465-780 nm.

3. The multiplex fluorescent staining method according to claim 2, characterized in that, The fluorochrome combination is selected from at least seven fluorochrome combinations formed by at least five fluorochromes with emission wavelengths of 465-700 nm.

4. The multiplex fluorescent staining method according to claim 3, characterized in that, The fluorochrome combination is selected from at least seven combinations of fluorochromes formed by at least five emission wavelengths selected from 465nm, 480nm, 490nm, 515nm, 520nm, 570nm, 620nm, 690nm, 700nm, and 780nm.

5. The multiplex fluorescent staining method according to claim 4, characterized in that, The test samples were stained sequentially using the following combination of fluorescein emission wavelengths: 480nm+520nm, 480nm+570nm, 480nm+620nm, 480nm+690nm, 520nm+570nm, 520nm+620nm, 520nm+690nm, 570nm+620nm, 570nm+690nm and 620nm+690nm.

6. The multiplex fluorescent staining method according to claim 5, characterized in that, The fluorophore was selected from the Opal 6-Plex Detection Kit for Whole Slide Imaging manufactured by Akoya Biosciences.

7. The multiplex fluorescent staining method according to claim 1, characterized in that, The fluorochrome combination is selected from at least five fluorochrome combinations selected from FITC, PE, APC, Alexa Fluor 647, Alexa Fluor 555, Alexa Fluor 488, FAM, 5-FAM, 5(6)-FAM, HEX, JOE, Lissamine Rhodamine B, Pyrene, ROX, TET, Tetramethyl Rhodamine, Texas Red, Dabcyl, CR-6G, BODIPY R6G, BODIPY FL, Amino-coumarin, Opal480Reagent, Opal 520Reagent, Opal570Reagent, Opal 620Reagent, and Opal 690Reagent.

8. The multiplex fluorescent staining method according to any one of claims 1-7, characterized in that, The multiplex fluorescent staining was performed using the Leica Bond RX platform.

9. The multiplex fluorescent staining method according to any one of claims 1-7, characterized in that, The multiplex fluorescent staining method includes the following steps performed sequentially: antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, incubation with the first fluorescein in the fluorescein combination, incubation with the second fluorescein in the same fluorescein combination; then, changing to another fluorescein combination, performing antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, and fluorescein incubation in sequence; until the target fluorescein is stained onto the sample to be stained; Preferably, the sample to be stained is a tissue section or a clinical smear.

10. The multiplex fluorescent staining method according to claim 9, characterized in that, The antigen retrieval refers to: treating the sample with an antigen retrieval solution and treating the dewaxed sample to be stained at 95-97℃ for 10-30 minutes. The incubation conditions for any one of the fluorescein combinations are: incubation at room temperature for 10 minutes; Preferably, incubate at 6℃-28℃ for 10 minutes.

Images