Multiplex fluorescence immunohistochemical staining kit and application

Abstract

The invention discloses a multiplex fluorescence immunohistochemical staining kit and application thereof. The multiplex fluorescence immunohistochemical staining kit comprises a plurality of casein amide substrate fluorescent dyes; the multi-tyramine substrate fluorescent dye comprises an aggregation-induced emission tyramine substrate and at least one narrow-spectrum tyramine substrate, the Stokes shift of the aggregation-induced emission tyramine substrate is larger than or equal to 150 nm, and the Stokes shift of the narrow-spectrum tyramine substrate is smaller than or equal to 60 nm. The full width at half maximum of the emission spectrum of the narrow-spectrum caseinate substrates is less than or equal to 50nm, and if the number of the narrow-spectrum caseinate substrates is more than or equal to two, the difference value of the maximum excitation wavelengths of any two narrow-spectrum caseinate substrates is more than or equal to 30nm. According to the present invention, the aggregation-induced emission tyrosine amide substrate with the large Stokes shift is used for the multiple fluorescence immunohistochemical staining, such that the staining multiple number is effectively improved, and the spectrum cross color does not occur;

Description

Technical Field

[0001] The invention belongs to the technical field of immunohistochemical detection, and in particular relates to a multiplex fluorescent immunohistochemical staining kit and application thereof. Background Art

[0002] In recent years, the treatment of malignant tumors has shifted from traditional surgery, radiotherapy, chemotherapy and other methods to immunotherapy. Immunotherapy is a breakthrough in traditional anti-cancer methods. Although immunotherapy has better specificity and durability than traditional therapies, its therapeutic effect is still deeply affected by the internal environment in which tumors and tumor cells live, that is, the physical and chemical and cytokine environment of the tumor microenvironment (TME). The tumor microenvironment (TME) is an important factor in tumor occurrence, development, response and anti-tumor treatment, and plays an important role in tumor prognosis and the effect of anti-tumor drugs. The complete tumor microenvironment includes extracellular matrix (ECM), soluble molecules and storm cells.

[0003] In recent years, it has been found in the study of tumor microenvironment that tumor infiltrating lymphocytes (TIL) are part of the monocytes infiltrating tumor tissues. They contain several subpopulations with different immunophenotypes and functions, such as CD8+ cytotoxic T cells, CD4+ helper T cells, B cells, and NK cells, and TILs are divided into TILs within the tumor-associated stroma, called sTILs, and TILs within tumor cell nests, called iTILs. Although TIL assessment has not yet been introduced into routine clinical / pathological practice, it is being integrated into biomarker discovery studies related to new immunotherapy trials and has the advantage of being able to quickly estimate tumor immune characteristics.

[0004] Tertiary lymphoid structures are a new hot spot in recent tumor immunotherapy. They are produced and exist near chronic inflammatory sites such as tumors, so they can more effectively inhibit related inflammatory responses than secondary lymphoid organs. Tertiary lymphoid structures are highly anticipated in tumor immunotherapy due to their direct location and powerful immune functions. They are composed of various types of immune cells, including antigen-specific B lymphocytes, T lymphocytes, and macrophages, which together drive immune responses against tumor development and progression.

[0005] The above-mentioned immune cells, macrophages and tumor cells express CD19 or CD20 on the surface of B cells through surface proteins, and T cells are divided into CD4+ effector T cells and CD8+ toxic T cells according to their expression of CD4 or CD8. Regulatory T cells express FOXP3+; CD68 is a typical surface marker protein of macrophages, and M2 macrophages express CD163. PanCK is a classic surface marker protein of tumor cells. At present, there is a lack of detection methods that can simultaneously study the number, location and spatial relationship of the above-mentioned immune cells and tumor cells, and explore the relationship between different combinations of biomarkers in the immune microenvironment and prognosis of patients with different cancer types.

[0006] In order to fully and accurately characterize the expression of the above-mentioned immune cells and their interactions in the immune microenvironment, it is necessary to detect these markers in situ at the same time. Although the existing multiple fluorescent immunohistochemistry kits can perform multi-label detection and analysis, the effects of these multiple immunohistochemistry staining kits are not the same. The selected substrate dyes are prone to spectral cross-coloring, which affects the analysis of the staining effect. In addition, the traditional staining method will affect the staining formed in the previous round during elution, and additional reagents such as staining enhancers are often required, and the test results are not accurate enough. In addition, due to the limited selection of spectral dyes, existing kits can often only support up to 6 markers for simultaneous labeling and staining. There is an urgent need to construct new dye systems and staining methods to achieve more multiple labeling and staining, so as to achieve more accurate analysis of the detection of different biomarkers in the immune microenvironment. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention aims to provide a multiplex fluorescent immunohistochemical staining kit and its application.

[0008] In view of the problems of spectral cross-talk and limited staining multiplicity in the existing multiple fluorescent immunohistochemical staining system, the present invention provides a multiple fluorescent immunohistochemical staining kit, which can realize the staining of multiple markers in the same tissue section with obvious staining contrast; for the problem that antibody elution affects the staining expression of the previous round of antibodies, the present invention proposes a multiple fluorescent immunohistochemical staining method, which does not affect the staining of the previous round of antibodies and has stable staining results.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions: The invention provides a multiple fluorescent immunohistochemical staining kit, comprising a plurality of tyramide substrate fluorescent dyes; the plurality of tyramide substrate fluorescent dyes comprise an aggregation-induced luminescence tyramide substrate and at least one narrow-spectrum tyramide substrate, the Stokes shift of the aggregation-induced luminescence tyramide substrate is greater than or equal to 150 nm, the Stokes shift of the narrow-spectrum tyramide substrate is less than or equal to 60 nm, the half-maximum full width of the emission spectrum of the narrow-spectrum tyramide substrate is less than or equal to 50 nm, and if there are more than or equal to two narrow-spectrum tyramide substrates, the difference between the maximum excitation wavelengths of any two narrow-spectrum tyramide substrates is greater than or equal to 30 nm.

[0010] The aggregation-induced luminescence tyramide substrate is a tyramide compound with an AIE fluorophore and a large Stokes shift, that is, a tyramide-labeled aggregation-induced luminescence molecule.

[0011] Preferably, the maximum excitation wavelength of the aggregation-induced luminescence tyramide substrate is 360 nm to 600 nm; that is, when the maximum excitation wavelength is 360 nm to 600 nm (for example, 360 nm, 400 nm, 430 nm, 450 nm, 480 nm, 510 nm, 550 nm or 600 nm), the maximum emission wavelength is 510 nm-800 nm (for example, 510 nm, 600 nm, 650 nm, 700 nm, 750 nm or 800 nm).

[0012] Further preferably, when the aggregation-induced luminescence tyramide substrate is used under a confocal microscope or a multispectral imager, the fluorescence wavelength can be set to: a fixed excitation wavelength of 440 nm and an emission wavelength of 590 nm to 650 nm.

[0013] Preferably, the structural formula of the aggregation-induced luminescence tyramide substrate is as follows: ;

[0014] R1, R2 and R3 are each independently selected from one of hydrogen, hydroxyl, cyano, methoxy, carboxyl and acetyl, Y is at least one of fluorine, chlorine, bromine and iodine; n is each independently a positive integer of 1 to 6. n is preferably a positive integer of 1 to 4 (1, 2, 3, 4).

[0015] Further preferably, the structural formula of the aggregation-induced luminescence tyramide substrate is as follows: ;

[0016] ;

[0017] ;

[0018] Preferably, the maximum excitation wavelength of the narrow spectrum tyramide substrate is 300nm~800nm; that is, when the maximum excitation wavelength is 300nm~800nm ​​(for example, 300nm, 360nm, 400nm, 430nm, 450nm, 480nm, 510nm, 550nm, 600nm, 650nm, 700nm, 750nm or 800nm), the maximum emission wavelength is 310nm~860nm (for example, 310nm, 350nm, 4000nm, 450nm, 510nm, 600nm, 650nm, 700nm, 750nm, 800nm ​​or 860nm); Preferably, the Stokes shift of the narrow spectrum tyramide substrate is 15 to 60 nm (15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm or 60 nm).

[0019] Preferably, the narrow spectrum tyramide substrate is a narrow spectrum dye molecule labeled with tyramide; the narrow spectrum dye molecule is a fluorescein dye molecule; Further preferably, the narrow spectrum dye molecule is selected from at least one of ATTO 425 (corresponding to TSA 480), Alexa 488 (corresponding to TSA 520), Cy3 (corresponding to TSA 570), Alexa 594 (corresponding to TSA 620), Alexa 633 (corresponding to TSA 650), Cy5, Cy5.5 (corresponding to TSA 690), and Cy7 (corresponding to TSA 780).

[0020] Preferably, the multiple tyramide substrate fluorescent dyes are dissolved in a buffer solution or an organic solvent, respectively.

[0021] Further preferably, the buffer is PBS or Tris-HCl, and its pH is 7.0-8.0.

[0022] Preferably, the working concentration of the aggregation-induced luminescence tyramide substrate is 100-1000 μM; Preferably, the working concentration of the narrow spectrum tyramide substrates is independently 1 to 20 μM.

[0023] Preferably, the multiplex fluorescent immunohistochemical staining kit further comprises an endogenous peroxidase blocker, a blocking solution, an antigen repair solution, a secondary antibody labeled with poly-HRP enzyme, a monoclonal antibody, a DAPI counterstain and a mounting medium.

[0024] Further preferably, the antigen repair solution is a 1-3 mM EDTA solution at pH 6.0-9.0 and a 10-20 mM sodium citrate repair solution at pH 3.0-6.0 containing 0.01-0.1 wt % Tween-20, or a 10-20 mM sodium citrate repair solution at pH 3.0-6.0 containing 0.01-0.1 wt % Tween-20; More preferably, the antigen repair solution is a 1 mM EDTA solution at pH 9.0 and a 10 mM sodium citrate repair solution at pH 6.0 containing 0.05 wt % Tween-20, or a 10 mM sodium citrate repair solution at pH 6.0 containing 0.05 wt % Tween-20; Further preferably, the endogenous peroxidase blocker is an aqueous solution containing 1-3 wt % hydrogen peroxide; More preferably, the endogenous peroxidase blocker is an aqueous solution containing 3 wt% (W / W) hydrogen peroxide; Further preferably, the blocking solution contains 0.01-0.02 M PBS and 2-5 wt % BSA; More preferably, the blocking solution contains 0.01 M PBS and 5 wt % BSA; Further preferably, the poly-HRP enzyme-labeled secondary antibody is a poly-HRP enzyme-labeled goat anti-mouse / rabbit IgG antibody; Further preferably, the monoclonal antibody is an immunohistochemical antibody corresponding to the marker; the monoclonal antibody is a mouse anti-monoclonal antibody or a rabbit anti-monoclonal antibody; More preferably, the marker is a marker associated with the immune microenvironment.

[0025] More preferably, the immune microenvironment-related markers are: B cell-related markers, such as CD19, CD20, CD21, CD23, etc.; T cell markers, such as CD3 (universal T cell marker), CD8 (cytotoxic T cell marker), CD4 (regulatory helper T cell marker), Foxp3 (regulatory T cell marker), CD45RO (memory T cell); macrophage-related markers, such as CD68 (generally refers to all macrophages), CD80, iNOS (M1 macrophages); CD206, CD204, CD163 (M2 macrophages); NK cell-related markers, For example, CD16, CD56, CD57, CD59, CD11b, CD94; tumor vascular endothelial cell markers, such as CD31, CD146, CD105; tumor fibroblast markers, such as α-SMA, FAP, S100A4; immune target markers, such as PD-1, PD-L1, CTLA-4, TIM3, LAG3; tumor cell markers, such as Pan-CK (pan-spectrum keratin) or EPCAM (epithelial cell adhesion molecule).

[0026] Most preferably, the markers are CD4, CD8, CD20, Foxp3, CD68, CD56, PD1, PD-L1, and CK.

[0027] Further preferably, the DAPI counterstain is an aqueous solution of 4',6-diamidino-2-phenylindole.

[0028] Further preferably, the sealing agent is an anti-fluorescence quenching agent.

[0029] The present invention provides an application of the above-mentioned multiple fluorescent immunohistochemical staining kit in immunohistochemical staining for non-disease diagnosis and treatment. The immunohistochemical staining method of the present invention is used in the field of non-disease diagnosis, such as multiple fluorescent immunohistochemical staining purely for scientific research.

[0030] Preferably, the immunohistochemical staining is used to detect markers of the tumor microenvironment.

[0031] The present invention provides a multiple fluorescent immunohistochemical staining method based on the multiple fluorescent immunohistochemical staining kit, comprising the following steps: (1) Pre-treating tissue sections; (2) Add endogenous peroxidase blocking treatment to the pretreated tissue sections and wash; (3) Then add blocking solution and incubate. After completion, aspirate or spin dry the liquid; (4) Then add a monoclonal antibody for incubation and wash; (5) Then add the secondary antibody labeled with poly-HRP enzyme for incubation and washing; (6) then adding a tyramide substrate fluorescent dye for incubation and staining, followed by washing; (7) Then add antigen repair solution and microwave antibody elution and wash; (8) Repeating steps (2) to (7) with different monoclonal antibodies and tyramide substrate fluorescent dyes until all monoclonal antibodies have bound to the tissue sections once, thereby obtaining a multi-labeled complex; (9) Finally, re-stain and seal the slides.

[0032] Preferably, the microwave antibody elution in step (7) is specifically as follows: Heat on medium-high heat for 6-8 minutes until the antigen retrieval solution (10-20 mM sodium citrate retrieval solution with pH 3.0-6.0 and 0.01-0.1 wt% Tween-20) boils, then turn off the heat and let it stand for 6-10 minutes, then turn to low heat for 4-8 minutes, then turn off the heat and let it stand for 4-8 minutes; Further preferably, the microwave antibody elution is specifically as follows: First heat on medium-high heat for 6-8 minutes until the antigen retrieval solution boils, then turn off the heat and let it sit for 8 minutes, then turn to low heat for 6 minutes, and then let it sit for 5 minutes.

[0033] Further preferably, the microwave power of the medium-high fire is 420W~560W; the microwave power of the low fire is 70W~210W.

[0034] Preferably, the washing in step (7) is to wait for the liquid in the pot to cool naturally to room temperature, take out the slices, remove the residual water on the slices, let them stand for 30 seconds, then put them into PBST and wash once, each time for 5 minutes.

[0035] Preferably, the tissue section pretreatment in step (1) includes baking, dewaxing and hydration, and antigen retrieval.

[0036] Further preferably, the baking is to place the paraffin sections face up in a constant temperature oven at 65-75°C (65°C, 68°C, 70°C, 73°C or 75°C) for 60-120 min (e.g., 60 min, 70 min, 80 min, 100 min, 110 min or 120 min), and then return to room temperature.

[0037] Further preferably, the dewaxing and hydrating is as follows: placing the baked paraffin sections in fresh dewaxing transparent liquid, soaking once, each time for 10 to 20 minutes, then changing to fresh dewaxing liquid and soaking once, each time for 10 to 20 minutes; then placing in anhydrous ethanol, soaking for 3 to 5 minutes; after removing excess liquid, placing in 95% ethanol, soaking for 3 to 5 minutes; then placing in 85% ethanol, soaking for 3 to 5 minutes; then placing in 75% ethanol, soaking for 3 to 5 minutes; finally placing in PBST (1×PBS phosphate buffer, containing 0.05% Tween-20) for rinsing for 3 to 5 minutes.

[0038] Further preferably, the antigen repair is as follows: placing the dewaxed and hydrated sections on a high temperature resistant staining rack, immersing the entire section in an antigen repair solution and placing the section in a microwave oven for heat repair of the antigen.

[0039] More preferably, the heat repair antigen is specifically as follows: first heat at medium-high heat for 6-8 minutes until the antigen repair solution (1mM EDTA solution at pH 9.0, or 10mM sodium citrate repair solution at pH 6.0 containing 0.05% Tween-20) boils, then turn off the heat and let it stand for 8 minutes, then switch to medium-low heat or low heat for 7 minutes, then turn off the heat and let it stand for 5 minutes. After the liquid in the pot cools naturally to room temperature, take out the slices, remove the residual water on the slices, draw circles around the tissue sample area with an immunohistochemistry waterproof pen, let it stand for 30 seconds, then put it in PBST and wash once, each time for 5 minutes.

[0040] Preferably, the endogenous peroxidase blocking treatment is added to the pretreated tissue slice in step (2) by dripping 3 (wt)% hydrogen peroxide aqueous solution into the central area of ​​the tissue slice and incubating at room temperature for 5 to 30 minutes (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes or 30 minutes).

[0041] Preferably, the operation of adding blocking solution and incubating in step (3) is: adding blocking solution containing 5% BSA to the central area of ​​the tissue section and incubating at room temperature for 10 to 30 minutes (for example, 10 minutes, 20 minutes or 30 minutes).

[0042] Preferably, when the monoclonal antibody in step (4) is CD68 monoclonal antibody, PD-L1 monoclonal antibody, PD1 monoclonal antibody, CD20 monoclonal antibody, CD4 monoclonal antibody, or CK monoclonal antibody, the incubation temperature is 35-42°C; when the monoclonal antibody to be incubated is CD56 monoclonal antibody, CD8 monoclonal antibody, or Foxp3 monoclonal antibody, the incubation temperature is 2-8°C.

[0043] Preferably, the monoclonal antibody in step (4) is used at a dilution factor of 50 to 600 times (e.g., 1:50, 1:100, 1:200, 1:300, 1:400 or 1:500).

[0044] Preferably, the step (5) of adding a poly-HRP enzyme-labeled secondary antibody for incubation is as follows: adding a poly-HRP enzyme-labeled secondary antibody solution and incubating at room temperature for 15 to 30 min (e.g., 15 min, 20 min, 25 min or 30 min).

[0045] Further preferably, the poly-HRP enzyme-labeled secondary antibody solution is a solution obtained by dissolving the poly-HRP enzyme-labeled secondary antibody in a buffer, and the buffer is PBS or Tris-HCl, and its pH is 7.0-8.0 (for example, 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0).

[0046] Further preferably, in the poly-HRP enzyme-labeled secondary antibody solution, the poly-HRP enzyme-labeled secondary antibody is dissolved in a buffer solution to form a ready-to-use working solution.

[0047] Preferably, the operation of adding a tyramide substrate fluorescent dye for incubation and staining in step (6) is: adding a tyramide substrate fluorescent dye solution and incubating at room temperature for 5 to 20 minutes (e.g., 5 minutes, 10 minutes, 15 minutes, 20 minutes).

[0048] Further preferably, the working concentration of the aggregation-induced luminescence tyramide substrate solution is 100-1000 μM, for example, 100 μM, 200 μM, 400 μM, 500 μM, 700 μM or 1000 μM.

[0049] Further preferably, the working concentration in the narrow spectrum tyramide substrate solution is 1 to 20 μM, for example 1 μM, 2 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 10 μM, 12 μM, 15 μM, 17 μM, 19 μM or 20 μM.

[0050] Any aggregation-induced luminescence tyramide substrate or narrow spectrum tyramide substrate solution (tyramide substrate fluorescent dye solution) is specifically prepared by adding the aggregation-induced luminescence tyramide substrate or narrow spectrum tyramide substrate mother solution (tyramide substrate fluorescent dye mother solution) into the signal amplification solution in proportion.

[0051] Further preferably, the signal amplification solution is a solution obtained by mixing a buffer solution, an organic phase solution, and a signal amplification substrate, the buffer solution is PBS or Tris-HCl, the pH of the buffer solution is 7.0-8.0, the volume content of the organic phase is 0-40% (for example, 0%, 1%, 10%, 15%, 20%, 30% or 40%), and the organic phase is dimethyl sulfoxide or N,N-dimethylformamide; the signal amplification substrate is hydrogen peroxide or urea peroxide, and the mass concentration of the signal amplification substrate is 0.001-0.01%.

[0052] Further preferably, the tyramide substrate fluorescent dye mother solution is a solution obtained by dissolving an aggregation-induced luminescence tyramide substrate or a narrow spectrum tyramide substrate in a buffer solution or an organic phase solution, and the buffer solution is PBS or Tris-HCl, and its pH is 7.0-8.0 (e.g., 7.0, 7.2, 7.4, 7.6, 7.8 or 8.0).

[0053] The operation of repeating steps (2) to (7) to obtain the multi-labeled complex is the same as described above.

[0054] Preferably, the counterstaining and sealing in step (9) are as follows: counterstaining the nucleus with a 4',6-diamidino-2-phenylindole (DAPI) aqueous solution for 5-10 min (e.g., 5 min, 6 min, 7 min, 8 min, 9 min or 10 min), then dripping an anti-fluorescence quenching sealing solution onto the tissue section, covering it with a coverslip, allowing the section to fully contact the sealing solution, and avoiding bubbles as much as possible.

[0055] Further preferably, the working concentration of the 4',6-diamidino-2-phenylindole aqueous solution (DAPI) is 0.5-10 μg / mL, for example, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL or 10 μg / mL.

[0056] As a preferred technical solution, the multiplex fluorescent immunohistochemistry method for tumor microenvironment detection of the present invention comprises the following steps: (1) Tissue section pretreatment; (2) Add 3 wt% hydrogen peroxide aqueous solution to the central area of ​​the tissue section to block endogenous peroxidase and incubate at room temperature for 5-30 min; (3) Add blocking solution containing 5 wt% BSA to the central area of ​​the tissue section and incubate at room temperature for 10-30 min; (4) Add a buffer solution of the monoclonal primary antibody of the tumor microenvironment to the pretreated tissue sections, with the volume ratio of the primary antibody to the buffer solution being 1:50-1000, and incubate at 35-42°C for 60 min or at 2-8°C overnight; (5) Add the buffer solution of poly-HRP enzyme-labeled secondary antibody to the slices after primary antibody incubation and incubate at room temperature in a wet box for 15-30 min. (6) adding any one of the aggregation-induced luminescence tyramide substrates or the narrow spectrum tyramide substrate solution, and incubating at room temperature for 5 to 20 minutes, wherein the concentration of the aggregation-induced luminescence tyramide substrate is 100 to 1000 μM; the concentration of the narrow spectrum tyramide substrate solution is 1 to 20 μM; (7) Place the sections after immunohistochemical staining on a high-temperature staining rack, immerse the whole section in the antigen repair solution and place it in a microwave oven for heat repair of antigens. Heat the sections on medium-high heat for 6-8 minutes until the antigen repair solution (10 mM sodium citrate repair solution with pH 6.0 and 0.05% Tween-20) boils, then turn off the heat and let it stand for 8 minutes, then turn to low heat for 6 minutes, and then let it stand for 5 minutes; (8) Repeat steps (2) to (7) to obtain a multi-labeled complex, the operation is the same as above; (9) Counterstain the nuclei with DAPI aqueous solution for 5–10 min, then drop anti-fluorescence quenching sealing solution on the tissue sections and cover with a coverslip.

[0057] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses multiple fluorescent immunohistochemical staining technology to detect relevant markers of tumor microenvironment through the combination of large Stokes shift aggregation-induced luminescence tyramide substrate and narrow spectrum tyramide substrate enzyme-catalyzed signal amplification technology. Compared with the existing multiple fluorescent immunohistochemical staining technology, the present invention can make full use of the spectral properties of different dyes, thereby expanding the number of detection multiplexities and is more conducive to clear in situ observation of immunochromogenic indicator markers.

[0058] The multiplex fluorescent immunohistochemical staining method of the present invention utilizes an improved antibody elution mode so that the staining effect of the previous round of antibodies is not affected by the subsequent microwave antibody elution operation, which is particularly important for ultra-multiple marker detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a seven-fold staining diagram for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I) in Example 1.

[0060] Figure 2This is an 8-fold staining diagram for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I) in Example 2.

[0061] Figure 3 This is a seven-fold staining diagram for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in lung cancer tissue using molecular formula (II) in Example 3.

[0062] Figure 4 This is an 8-fold staining diagram for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in lung cancer tissue using molecular formula (II) in Example 4.

[0063] Figure 5 This is a combined diagram of single staining and elution conditions of the FoxP3 antibody in Example 5.

[0064] Figure 6 This is a combined diagram of single staining and elution conditions for the CD4 antibody of Example 5.

[0065] Figure 7 This is a combined diagram of single staining and elution conditions for the PD1 antibody of Example 5.

[0066] Figure 8 This is the double staining combination and single channel image of CD4-CD8 and AIE compound (a) of Comparative Example 1.

[0067] Fig. 9 This is the double staining combination and single channel image of CD4-CD8 and AIE compound (ii) of Comparative Example 1.

[0068] Fig.10 This is the double staining combination and single channel image of CD4-CD8 and AIE compound (III) of Comparative Example 1.

[0069] Fig.11 This is the double staining combination and single channel image of CD4-CD8 and AIE compound (IV) of Comparative Example 1.

[0070] Fig.12 This is a combined diagram of single staining and elution conditions of the FoxP3 antibody in comparative example 2.

[0071] Fig.13 It is a combined diagram of single staining and elution conditions of the CD4 antibody in comparative example 2.

[0072] Fig.14 This is a combined diagram of single staining and elution conditions for the PD1 antibody of comparative example 2. DETAILED DESCRIPTION

[0073] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

[0074] The molecular structure of the AIE tyramide substrate is as follows: ;

[0075] ;

[0076] Embodiment 1: The seven-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I) specifically comprises the following steps: 1. Baking: slice the paraffin tissue (75 mm 25mm) pieces are placed upwards and baked in a constant temperature oven at 70℃ for 120min, and then slowly returned to room temperature.

[0077] 2. Dewaxing and hydration: Place the paraffin sections in fresh dewaxing transparent liquid - xylene for 20 minutes / time, then change to fresh xylene for 15 minutes / time; then place them in anhydrous ethanol and soak for 3 minutes; after removing the excess liquid, place them in 95% ethanol and soak for 3 minutes; then place them in 85% ethanol and soak for 3 minutes; then place them in 75% ethanol and soak for 3 minutes; rinse them in PBST (1×PBS buffer, containing 0.05% Tween-20) for 3 minutes.

[0078] 3. Antigen repair: Soak the whole slice in antigen repair solution (1mM EDTA solution with pH 9.0), heat for 6~8min at medium-high heat (80% power, maximum microwave power 700W) until the antigen repair solution (1mM EDTA solution with pH 9.0) boils, then turn off the heat and let it stand for 8min, then switch to medium-low or low heat (30%~40% power) for 7min, then turn off the heat and let it stand for 5min. Wait for the liquid in the pot to cool naturally to room temperature, take out the slice, remove the residual water on the slice, draw a circle around the tissue sample area with an immunohistochemical waterproof pen, let it stand for 30s, then put it in PBST, wash once, 5 minutes each time.

[0079] 4. Blocking of endogenous peroxidase sites: Add 100 μL of endogenous peroxidase blocking solution (3% hydrogen peroxide aqueous solution, homemade) to the tissue area of ​​the slice, and incubate in a wet box at room temperature for 15 minutes. After completion, place in PBST and wash three times, each time for 3 minutes.

[0080] 5. Non-specific site blocking: Add 100 μL of 5% BSA solution (1× PBS solution containing 5% BSA) to the tissue area of ​​the slice, incubate in a wet box at room temperature for 30 minutes to block non-specific sites. After completion, try to absorb or dry the liquid.

[0081] 6. Primary antibody incubation: Add 100 μL of Foxp3 monoclonal antibody working solution to the tissue area of ​​the slice, with a dilution ratio of 1:100 (diluent is 1× PBS), and incubate overnight at 4°C for 16 hours. After incubation, wash with PBST three times, 5 minutes each time.

[0082] 7. Secondary antibody incubation: Add 50-100 μL of poly-HRP-labeled goat anti-mouse / rabbit IgG antibody working solution to the tissue area of ​​the slice, incubate at room temperature for 20 minutes, and wash with PBST three times after incubation, each time for 5 minutes.

[0083] 8. Fluorescence staining: Add 100 μL of TSA 620 substrate working solution to the tissue area of ​​the slice (TSA 620 mother solution and signal amplification solution are diluted proportionally to a working solution with a substrate concentration of 10 μM. The components of the signal amplification solution are: 0.1 M Tris-HCL buffer, containing 0% volume concentration of DMSO and 0.002% mass concentration of hydrogen peroxide. This signal amplification solution is suitable for all subsequent fluorescent substrates), incubate at room temperature for 10 minutes, and wash with PBST three times after incubation, each time for 5 minutes.

[0084] 9. Antibody elution: Place the slices in antigen repair solution (10mM sodium citrate repair solution with pH 6.0 and 0.05% Tween-20), heat for 6-8 minutes at medium-high heat (80% power, microwave oven maximum power 700W) until the antigen repair solution (10mM sodium citrate repair solution with pH 6.0 and 0.05% Tween-20) boils, then turn off the heat and let it stand for 8 minutes, then turn to low heat (30% power) for 6 minutes, then turn off the heat and let it stand for 5 minutes. Wait for the liquid in the pot to cool naturally to room temperature, take out the slices, remove the residual water on the slices, let it stand for 30 seconds, then put it in PBST and wash once, 5 minutes each time.

[0085] 10. Antibody staining in sequence: After the first antibody staining is completed, steps 4 to 9 need to be repeated for each subsequent antibody, and all antibodies are labeled in sequence to obtain a multi-labeled complex.

[0086] The labeling order of the monoclonal antibodies is: FoxP3 antibody, CD4 antibody, CD68 antibody, CD20 antibody, PD1 antibody, PD-L1 antibody, CK antibody, and the substrate working solution is shown in Table 1.

[0087] 11. Add DAPI for counterstaining: add 100 μL of DAPI solution to the tissue area of ​​the slice, the working concentration of DAPI (purchased from Shanghai Bio-Tech Biotechnology, model: C1002) is 5 μg / mL, counterstain the nucleus for 7 minutes, and wash twice with PBST, each time for 5 minutes.

[0088] 12. Anti-fluorescence quenching agent sealing: Add 100 μL of anti-fluorescence quenching sealing solution (purchased from Haosai Tuohua, product number: CMT110) to the tissue area of ​​the slice, cover with a coverslip, let the slice fully contact the sealing solution, and try to avoid bubbles.

[0089] 13. Microscopic observation and photography: Use Leica confocal fluorescence microscope or Akoya's Vectra system to collect continuous spectra and perform image processing.

[0090] The steps of adding reagents can be summarized as shown in Table 1: Table 1

[0091] Among them, the dye spectrum information of the multicolor substrate is shown in Table 2: Table 2

[0092] It can be seen that the kit of this embodiment can simultaneously label up to 7 different antigens on the same slice, and the multi-spectral imaging system with spectral splitting function and Akoya's inForm software are used for spectral splitting (after obtaining the λ-Stack spectral intensity curve data by continuous spectral imaging of samples with single fluorescent staining, the signal intensity is split in corresponding proportions for the fluorescent channels with the same spectral characteristics based on the splitting algorithm), which can achieve accurate imaging of 7-fold 8-color staining (see the results). Figure 1 ).

[0093] Embodiment 2: The eight-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I) has the same specific operation steps as Example 1. The specific staining rounds and primary antibody and substrate reagent information are shown in Table 3: Table 3

[0094] Among them, the dye spectrum information of the multicolor substrate is shown in Table 4: Table 4

[0095] It can be seen that the kit of this embodiment can simultaneously label up to 8 different antigens on the same slice, and the multi-spectral imaging system with spectral separation function can accurately separate and image 8-fold 9-color staining (see the results). Figure 2 ).

[0096] Embodiment 3: The seven-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in lung cancer tissue using molecular formula (II) has the same specific operation steps as those in Example 1 above. The specific staining rounds and primary antibody and substrate reagent information are shown in Table 5: Table 5

[0097] Among them, the dye spectrum information of the multicolor substrate is shown in Table 6: Table 6

[0098] It can be seen that the kit of this example can simultaneously label up to 7 different antigens on the same slice. Relying on the multi-spectral imaging system with spectral splitting function and using Akoya's inForm software for spectral splitting, it can achieve accurate imaging of 7-fold 8-color staining (see the results). Figure 3 ).

[0099] Embodiment 4: The eight-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (II) has the same specific operation steps as Example 1. The specific staining rounds and primary antibody and substrate reagent information are shown in Table 7: Table 7

[0100] It can be seen that the kit of this example can simultaneously label up to 8 different antigens on the same slice. Relying on the multispectral imaging system with spectral splitting function and using Akoya's inForm software for spectral splitting, it can achieve accurate imaging of 8-fold 9-color staining (see the results). Figure 4 ).

[0101] Embodiment 5: In order to test the antibody elution method, single staining was used to explore the microwave treatment antibody elution method, including microwave treatment time, selection of repair solution, etc. The specific treatment conditions are as follows: 1. Baking the slices: The specific steps are the same as those in Example 1.

[0102] 2. Dewaxing and hydration: The specific steps are the same as those in Example 1.

[0103] 3. Antigen retrieval: The specific steps are the same as those in Example 1.

[0104] 4. Endogenous peroxidase site blocking: The specific steps are the same as those in Example 1.

[0105] 5. Non-specific site blocking: The specific steps are the same as those in Example 1.

[0106] 6. Primary antibody incubation: Three marker antibodies were selected from tumor microenvironment-related markers for verification, namely FoxP3, CD4, and PD1. The primary antibody treatment and incubation were the same as in Example 1.

[0107] 7. Secondary antibody incubation: The specific steps are the same as those in Example 1.

[0108] 8. Secondary antibody elution treatment: Set different conditions by adjusting the microwave treatment time and repair solution. The specific information is shown in Table 8: Table 8

[0109] 9. Add TSA570 substrate for staining: The specific steps are the same as those in Example 1.

[0110] 10. Add DAPI for counterstaining: The specific steps are the same as those in Example 1.

[0111] 11. Anti-fluorescence quenching agent sealing: The specific steps are the same as those in Example 1.

[0112] Observation was performed by confocal microscopy / fluorescence microscopy (see the results). Figure 5-Figure 7 ), and compared with comparative example 2, it was found that through the improved microwave treatment method, no obvious positive staining phenomenon was observed under the three elution times, whether in citric acid repair solution or EDTA repair solution, and only DAPI nuclear staining signal was seen, indicating that the primary antibody-secondary antibody complex can be completely eluted by microwave treatment under this elution mode, and the minimum total elution time can also be applied to the elution method in multiple fluorescent immunohistochemistry staining.

[0113] Embodiment 6: In order to test the effect of the improved elution method on the antibody after multiple rounds of elution, low-abundance expressed markers were selected, stained normally with single staining, and then eluted for different times. The specific processing conditions are as follows: 1. Baking the slices: The specific steps are the same as those in Example 1.

[0114] 2. Dewaxing and hydration: The specific steps are the same as those in Example 1.

[0115] 3. Antigen retrieval: The specific steps are the same as those in Example 1.

[0116] 4. Endogenous peroxidase site blocking: The specific steps are the same as those in Example 1.

[0117] 5. Non-specific site blocking: The specific steps are the same as those in Example 1.

[0118] 6. Primary antibody incubation: Two low-abundance marker antibodies were selected from tumor microenvironment-related markers for verification, namely FoxP3 and CD56. The primary antibody treatment and incubation were the same as in Examples 1 and 2.

[0119] 7. Secondary antibody incubation: The specific steps are the same as those in Example 1.

[0120] 8. Add TSA570 substrate for staining: The specific steps are the same as those in Example 1.

[0121] 9. Antibody complex elution treatment: By adjusting different elution treatment times to simulate multiple rounds of elution in multiple fluorescent immunohistochemistry, different conditions can be set. The specific information is shown in Table 9: Table 9

[0122] 10. Add DAPI for counterstaining: The specific steps are the same as those in Example 1.

[0123] 11. Anti-fluorescence quenching agent sealing: The specific steps are the same as those in Example 1.

[0124] Observation was performed using a confocal microscope / fluorescence microscope (results are shown in Table 10), and compared with Comparative Example 3, it was found that different rounds of elution were performed in a citric acid repair solution containing Tween-20 through the improved microwave treatment method. It was found that after 8 rounds of repair and elution, the antibody staining signal remained the same as before elution, but in the EDTA repair solution, the antibody staining signal gradually decreased after 8 rounds of repair and elution, indicating that EDTA was too strong when used as an elution repair solution, which was prone to excessive elution, indicating that the citric acid repair solution containing Tween-20 in this elution mode can be used as the main elution solution type, and at the same time, the effect of Tween-20 in the citric acid repair solution on the repair solution was discussed. After comparison, it was found that the citric acid repair solution containing Tween-20 basically maintained a stable fluorescence intensity in multiple rounds of repair, while the citric acid repair solution without Tween-20 had a slightly reduced fluorescence intensity after multiple rounds of repair, indicating that the citric acid repair solution (containing Tween-20) is suitable for the main elution repair solution under the elution method in multiple fluorescent immunohistochemical staining.

[0125] Table 10

[0126] Comparative Example 1: In order to verify the feasibility of AIE tyramide compounds in multicolor labeling, so that they do not cause cross-color interference with adjacent spectral dyes, the Stokes shift of each compound was used for verification and selection, and finally TSA 480 or TSA520 was used to double-stain with each compound to verify cross-color interference. The specific operation steps are the same as those in Example 1. The specific staining rounds and primary antibody and substrate reagent information are shown in Table 11: Table 11

[0127] Among them, the dye spectrum data and usage concentration of the AIE compound are shown in Table 12: Table 12

[0128] It can be seen that through the compound screening of this comparative example, the adjacent narrow spectrum dyes are used for double staining imaging (results are shown in Figure 8-Figure 11 ), it can be found that when double staining with AIE compound (I) and TSA 480, AIE compound (II) and TSA 480, CD8 shows single channel imaging staining and double staining synthesis Figure 1 The results show that the cross-talk between compounds (I) and (II) and TSA 480 is small, while when AIE compound (III) and TSA 520, AIE compound (IV) and TSA 480 are used for double staining, it is found that there is partial cross-talk between AIE compound (III) and TSA 520, which is manifested in that the CD8 staining signal is partially interfered by CD4; there is also slight cross-talk between AIE compound (IV) and TSA 480. AIE compounds (III) and (IV) cannot be combined with other dyes for multiple imaging during multiple staining, while AIE compounds (I) and (II) have little cross-talk with adjacent spectral dyes and can be used as multiple staining for labeling imaging. This shows that when the Stokes shift is ≥150nm, the cross-talk interference of dyes is small and multiple labeling imaging can be performed.

[0129] Comparative Example 2: Compared with the existing commonly used elution methods, the microwave treatment antibody elution method was explored using single staining, including microwave treatment time, selection of repair solution, etc. The specific treatment conditions are as follows: 1. Baking the slices: The specific steps are the same as those in Example 1.

[0130] 2. Dewaxing and hydration: The specific steps are the same as those in Example 1.

[0131] 3. Antigen retrieval: The specific steps are the same as those in Example 1.

[0132] 4. Endogenous peroxidase site blocking: The specific steps are the same as those in Example 1.

[0133] 5. Non-specific site blocking: The specific steps are the same as those in Example 1.

[0134] 6. Primary antibody incubation: Three marker antibodies were selected from tumor microenvironment-related markers for verification, namely FoxP3, CD4, and PD1. The primary antibody treatment and incubation were the same as in Example 1.

[0135] 7. Secondary antibody incubation: The specific steps are the same as those in Example 1.

[0136] 8. Secondary antibody elution treatment: Different conditions are set by adjusting the microwave treatment time and repair solution. The specific information is shown in Table 13: Table 13

[0137] 9. Add TSA570 substrate for staining: The specific steps are the same as those in Example 1.

[0138] 10. Add DAPI for counterstaining: The specific steps are the same as those in Example 1.

[0139] 11. Anti-fluorescence quenching agent sealing: The specific steps are the same as those in Example 1.

[0140] Observation was performed by confocal microscopy / fluorescence microscopy (see the results). Figure 12-14 ), and compared with Example 5, it was found that under this microwave treatment method, no obvious fluorescent staining signal remained on the slice at the 20 min elution time, indicating that the primary antibody-secondary antibody complex can also be eluted by microwave treatment under this elution mode, but considering the subsequent use of multiple rounds of repair treatment, this elution mode prolongs the time of maintaining medium and low heat, increases the elution intensity, and easily causes excessive antigen repair, and is not suitable for the elution method in multiple fluorescent immunohistochemical staining.

[0141] Comparative Example 3: In order to compare the effects of existing elution methods on antibodies after multiple rounds of elution, low-abundance expressed markers were selected, and normal staining was performed using single staining. Then, different elution times were performed with reference to Comparative Example 2. The specific treatment conditions are as follows: 1. Baking the slices: The specific steps are the same as those in Example 1.

[0142] 2. Dewaxing and hydration: The specific steps are the same as those in Example 1.

[0143] 3. Antigen retrieval: The specific steps are the same as those in Example 1.

[0144] 4. Endogenous peroxidase site blocking: The specific steps are the same as those in Example 1.

[0145] 5. Non-specific site blocking: The specific steps are the same as those in Example 1.

[0146] 6. Primary antibody incubation: Two low-abundance marker antibodies were selected from tumor microenvironment-related markers for verification, namely FoxP3 and CD56. The primary antibody treatment and incubation were the same as in Examples 1 and 2.

[0147] 7. Secondary antibody incubation: The specific steps are the same as those in Example 1.

[0148] 8. Add TSA570 substrate for staining: The specific steps are the same as those in Example 1.

[0149] 9. Antibody complex elution treatment: By adjusting different elution treatment times to simulate multiple rounds of elution in multiple fluorescent immunohistochemistry, different conditions can be set. The specific information is shown in Table 14: Table 14

[0150] 10. Add DAPI for counterstaining: The specific steps are the same as those in Example 1.

[0151] 11. Anti-fluorescence quenching agent sealing: The specific steps are the same as those in Example 1.

[0152] Observation by confocal microscopy / fluorescence microscopy (results in Table 15) revealed that the microwave treatment resulted in a decrease in staining intensity with increasing elution rounds in both citric acid repair solution and EDTA repair solution, indicating that the citric acid and EDTA used as elution repair solutions were too strong under this elution mode and could easily cause over-elution, indicating that this elution mode is not suitable for elution in multiple fluorescent immunohistochemical staining.

[0153] Table 15

[0154] The results of the embodiments and comparative examples are analyzed: Example 1 is a seven-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I), from Figure 1 It can be seen that the combination of aggregation-induced emission tyramide substrate and narrow spectrum dye can achieve the detection of seven markers of tumor microenvironment.

[0155] Example 2 is an 8-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (I), from Figure 2 It can be seen that the combination of aggregation-induced luminescence tyramide substrate and narrow spectrum dye can realize the detection of 8 markers of tumor microenvironment, effectively expanding the number of detection multiplexes.

[0156] Example 3 is a seven-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (II), from Figure 3 It can be seen that the combination of aggregation-induced emission tyramide substrate and narrow spectrum dye can achieve the detection of seven markers of tumor microenvironment.

[0157] Example 4 is an 8-fold staining method for multiple fluorescent immunohistochemical detection of tumor microenvironment-related markers in tonsil tissue using molecular formula (II), from Figure 4 It can be seen that the combination of aggregation-induced luminescence tyramide substrate and narrow spectrum dye can realize the detection of 8 markers of tumor microenvironment, effectively expanding the number of detection multiplexes.

[0158] Example 5 is a single staining to explore the microwave treatment antibody elution method, from Figure 5 , Figure 6 , Figure 7 It can be seen that no obvious positive staining phenomenon was observed under the three elution times, and only DAPI nuclear staining signals were seen, indicating that the primary antibody-secondary antibody complex can be completely eluted by microwave treatment under this elution mode, and the minimum total elution time can also be applied to the elution method in multiple fluorescent immunohistochemical staining.

[0159] Example 6 is a method for testing the effect of the improved elution method on the antibody after multiple rounds of elution. From Table 10, it is found that after 8 rounds of elution with citric acid repair solution, the antibody staining signal remains the same as before elution, but in EDTA repair solution, after 8 rounds of repair elution, the antibody staining signal gradually decreases, indicating that EDTA is too strong when used as an elution repair solution, which can easily cause excessive elution, indicating that in this elution mode, citric acid repair solution can be used as the main elution solution type, which is suitable for the elution method in multiple fluorescent immunohistochemistry staining.

[0160] Comparative Example 1 is to select AIE compound (I), AIE compound (II), AIE compound (III), and AIE compound (IV) to verify the screening method of double dyeing of compounds with different Stokes shift spectral characteristics with adjacent narrow spectrum dyes (TSA 480, TSA 520), from Figure 8 , Fig. 9 It can be seen that when using compound (I) and compound (II), the single channel image of CD8 in double staining is consistent with the imaging results in the combined image, indicating that the compound and the dye do not produce cross-color interference; Fig.10 , Fig.11 It can be seen that when using compound (III) and compound (IV), the imaging results of CD8 in the single channel image in double staining are different from those in the combined image. CD8 imaging is interfered by the CD4 staining signal, indicating that the compound and the dye produce cross-color interference and are not suitable for multiple staining labeling.

[0161] Comparative Example 2 is a commonly used elution method, using single staining to explore the microwave treatment antibody elution method. Fig.12 , Fig.13 , Fig.14It can be seen that the primary antibody-secondary antibody complex can also be eluted by microwave treatment in this elution mode. However, considering the subsequent use of multiple rounds of repair treatment, this elution mode prolongs the time of maintaining medium and low heat, increases the elution intensity, and easily causes excessive antigen repair. It is not suitable for the elution method in multiple fluorescent immunohistochemistry staining.

[0162] Comparative Example 3 is a single staining test method that compares the effects of existing elution methods on antibodies after multiple rounds of elution. It can be found from Table 15 that the staining intensity decreases with the increase in elution rounds in both the citric acid repair solution and the EDTA repair solution through this microwave treatment method, indicating that the citric acid and EDTA are too strong when used as elution repair solutions under this elution mode, which can easily cause excessive elution, indicating that this elution mode is not suitable for the elution method in multiple fluorescent immunohistochemistry staining.

[0163] In the present invention, multiple fluorescent immunohistochemical staining technology is used to detect relevant markers of tumor microenvironment through the combination of aggregation-induced luminescence tyramide substrate and enzyme-catalyzed signal amplification technology of narrow-spectrum tyramide substrate. Compared with the existing multiple fluorescent immunohistochemical staining technology, the staining method using the above embodiment can make full use of the spectral properties of different dyes, thereby expanding the number of detection multiplexes, and is more conducive to clear in situ observation of immunochromogenic indicator markers. In addition, the staining method utilizes an improved antibody elution mode, so that the staining effect of the previous round of antibodies is not affected by the subsequent microwave antibody elution operation.

[0164] The present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the compounds selected by the present invention, addition of auxiliary components in the application method system, selection of specific methods, etc., all fall within the protection scope of the present invention.

Claims

1. A multiplex fluorescent immunohistochemical staining kit, characterized in that: The invention comprises a plurality of tyramide substrate fluorescent dyes; the plurality of tyramide substrate fluorescent dyes comprise an aggregation-induced luminescence tyramide substrate and at least one narrow spectrum tyramide substrate, the Stokes shift of the aggregation-induced luminescence tyramide substrate is greater than or equal to 150 nm, the Stokes shift of the narrow spectrum tyramide substrate is less than or equal to 60 nm, the half-maximum full width of the emission spectrum of the narrow spectrum tyramide substrate is less than or equal to 50 nm, and if there are more than or equal to two narrow spectrum tyramide substrates, the difference between the maximum excitation wavelengths of any two narrow spectrum tyramide substrates is greater than or equal to 30 nm.

2. The multiplex fluorescent immunohistochemical staining kit according to claim 1, characterized in that: The maximum excitation wavelength of the aggregation-induced luminescence tyramide substrate is 360nm~600nm; The structural formula of the aggregation-induced luminescence tyramide substrate is as follows: R1, R2 and R3 are each independently selected from one of hydrogen, hydroxyl, cyano, methoxy, carboxyl and acetyl; Y is at least one of fluorine, chlorine, bromine and iodine; and n is each independently a positive integer of 1 to 6.

3. The multiplex fluorescent immunohistochemical staining kit according to claim 2, characterized in that: The structural formula of the aggregation-induced luminescence tyramide substrate is as follows: 。 4. The multiplex fluorescent immunohistochemical staining kit according to claim 1, characterized in that: The narrow spectrum tyramide substrate is a narrow spectrum dye molecule labeled with tyramide; the narrow spectrum dye molecule is selected from at least one of ATTO 425, Alexa488, Cy3, Alexa 594, Alexa 633, Cy5, Cy5.5, and Cy7.

5. The multiplex fluorescent immunohistochemical staining kit according to claim 1, characterized in that: The multiple tyramide substrate fluorescent dyes are respectively dissolved in a buffer solution or an organic solvent; The working concentration of the aggregation-induced luminescence tyramide substrate is 100-1000 μM; The working concentrations of the narrow spectrum tyramide substrates are independently 1-20 μM.

6. The multiplex fluorescent immunohistochemical staining kit according to claim 1, characterized in that: The multiple fluorescent immunohistochemical staining kit also includes an endogenous peroxidase blocker, a blocking solution, an antigen repair solution, a secondary antibody labeled with poly-HRP enzyme, a monoclonal antibody, a DAPI counterstain and a mounting medium.

7. The multiplex fluorescent immunohistochemical staining kit according to claim 6, characterized in that: The antigen repair solution is a 1-3 mM EDTA solution at pH 6.0-9.0 and a 10-20 mM sodium citrate repair solution at pH 3.0-6.0 containing 0.01-0.1 wt % Tween-20, or a 10-20 mM sodium citrate repair solution at pH 3.0-6.0 containing 0.01-0.1 wt % Tween-20; The endogenous peroxidase blocker is an aqueous solution containing 1-3 wt% hydrogen peroxide; The blocking solution contains 0.01-0.02M PBS and 2-5 wt% BSA; The poly-HRP enzyme-labeled secondary antibody is a poly-HRP enzyme-labeled goat anti-mouse / rabbit IgG antibody; The monoclonal antibody is an immunohistochemical antibody corresponding to the marker; the monoclonal antibody is a mouse anti-monoclonal antibody or a rabbit anti-monoclonal antibody; The DAPI counterstain is an aqueous solution of 4',6-diamidino-2-phenylindole; The sealing agent is an anti-fluorescence quenching agent.

8. Use of the multiplex fluorescent immunohistochemical staining kit according to any one of claims 1 to 7 in immunohistochemical staining for non-disease diagnosis and treatment.

9. A multiple fluorescent immunohistochemical staining method based on the multiple fluorescent immunohistochemical staining kit according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Pre-treating tissue sections; (2) Add endogenous peroxidase blocking treatment to the pretreated tissue sections and wash; (3) Then add blocking solution and incubate. After completion, aspirate or spin dry the liquid; (4) Then add a monoclonal antibody for incubation and wash; (5) Then add the secondary antibody labeled with poly-HRP enzyme for incubation and washing; (6) then adding a tyramide substrate fluorescent dye for incubation and staining, followed by washing; (7) Then add antigen repair solution and microwave antibody elution and wash; (8) Repeating steps (2) to (7) with different monoclonal antibodies and tyramide substrate fluorescent dyes to obtain a multi-labeled complex; (9) Finally, re-stain and seal the slides.

10. The multiplex fluorescent immunohistochemical staining method according to claim 9, characterized in that: The microwave antibody elution described in step (7) is specifically as follows: Heat on medium-high heat for 6-8 minutes until the antigen retrieval solution boils, then turn off the heat and let it stand for 6-10 minutes, then turn to low heat for 4-8 minutes, then turn off the heat and let it stand for 4-8 minutes; The incubation time in step (5) is 15 to 30 min; The incubation time in step (6) is 5 to 20 minutes.

Images