A multiple fluorescent staining kit for tissue samples

Abstract

This invention relates to the field of biodetection technology, specifically disclosing a multiplex fluorescent staining kit for tissue samples. The kit includes an antibody dissociation buffer, a signal amplification deposition solution, a fluorescent labeling reagent, a tissue-protective imaging medium, and an autofluorescence background inhibitor. This invention utilizes a non-destructive elution technique achieved through the synergistic effect of a low-pH buffer and a directional electric field. It leverages the positively charged nature of antibodies under acidic conditions to drive them away from the tissue and towards the negative electrode, achieving gentle and efficient antibody removal. Simultaneously, it employs a two-step cascade amplification strategy of small molecule deposition and copper-free click chemical labeling, balancing deep penetration with ultra-high sensitivity. This invention can stably support 6-12 cycles of staining, exhibits high elution efficiency, preserves tissue morphology, and can detect low-abundance targets. It is suitable for spatial analysis of the tumor microenvironment, monitoring of transplant immune rejection, and pathological evaluation of neurodegenerative diseases, demonstrating promising clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more specifically, to a multiplex fluorescent staining kit for tissue samples. Background Technology

[0002] Multiplex immunofluorescence (mIHC) is a key tool for analyzing the spatial heterogeneity of the tissue microenvironment, playing an irreplaceable role, especially in tumor immunoassay and neuropathology research. Existing multiplex staining strategies mainly fall into two categories: direct co-staining based on primary antibodies from different species, and cyclic staining based on tyramine signal amplification (TSA). However, both of these techniques face significant technical bottlenecks in achieving ultra-multiplex (>6 colors) detection.

[0003] Direct co-staining methods are limited by overlapping fluorescence spectra and the scarcity of primary antibody host species, typically making it difficult to simultaneously detect more than 4-5 targets on the same slide, thus failing to meet the analytical needs of complex microenvironments. While cyclic staining techniques overcome species limitations through a "staining-imaging-elution" cycle, their core challenge lies in the antibody elution step. Current mainstream elution protocols primarily rely on high-temperature microwave retardation or strong chemical reagents (such as high-concentration SDS, DTT reducing agent, and prolonged immersion in low-pH glycine). High-temperature treatment easily leads to tissue morphology collapse, irreversible denaturation of antigenic epitopes, and slide detachment; strong reducing agents disrupt the disulfide bond structure of tissue proteins, resulting in antigen loss in subsequent rounds. Furthermore, traditional elution relies on passive diffusion, which is inefficient and prone to residue, causing signal crosstalk between rounds. It is noteworthy that electric field-driven technology has been widely used in nucleic acid electrophoresis and protein separation, but its application to the targeted removal of antibodies from tissue slides has not yet been reported. The main challenge lies in achieving efficient removal without damaging the tissue and avoiding the disruption caused by electrolytic bubbles.

[0004] In the signal amplification stage, TSA technology uses small-molecule tyramine deposition, which, although highly permeable, has limited single-point signal gain, making it difficult to detect low-abundance targets such as PD-L1. If pre-polymerized macromolecular probes are used to enhance the signal, steric hindrance makes it difficult to penetrate deep into dense tissues, leading to uneven labeling. In recent years, click chemistry has been widely used for biomolecular labeling due to its high efficiency and orthogonal reaction characteristics. However, the traditional copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) requires copper ion catalysis. The reactive oxygen species induced by copper ions not only quench the fluorescence signal but also cause oxidative damage to tissue samples, limiting its application in multi-cycle staining of tissue sections. Based on the above, this invention proposes a multiplex fluorescent staining kit for tissue samples. Summary of the Invention

[0005] To address the problems of tissue damage caused by harsh antibody elution conditions in existing technologies and the difficulty in balancing sensitivity and permeability in traditional signal amplification methods, this invention provides a multiplex fluorescent staining kit for tissue samples.

[0006] In a first aspect, the present invention provides a multiplex fluorescent staining kit for tissue samples, comprising the following components: antibody dissociation buffer, signal amplification deposition solution, fluorescent labeling reagent, tissue protective imaging medium, and autofluorescence background inhibitor.

[0007] Preferably, the antibody dissociation buffer is an aqueous solution containing 50-200 mM glycine, 300-500 mM sodium chloride and 0.05-0.2 wt% Tween-20, with a pH of 2.2-3.0.

[0008] Preferably, the signal amplification deposition solution is an aqueous solution containing 5-20 μg / mL of HRP catalytic substrate, wherein the HRP catalytic substrate is a compound containing both a tyrosine structure and a first click chemical reaction functional group; the first click chemical reaction functional group is cyclooctyne.

[0009] Preferably, the fluorescent labeling reagent is an aqueous solution containing 2-10 μg / mL of a fluorescent dye coupled with a second click chemistry functional group; the second click chemistry functional group is an azide group; and the fluorescent dye is selected from one or more of AF488, AF555, AF594, AF647, AF680 and AF750.

[0010] Preferably, the tissue-protective imaging medium is composed of 1-3 wt% aqueous methylcellulose solution, 0.1-0.5 wt% aqueous vitamin E derivative solution, and PBS buffer in a volume ratio of 1:1:6-10.

[0011] Preferably, the vitamin E derivative is selected from one or more of sodium vitamin E phosphate, polyethylene glycol succinate, and sodium vitamin E succinate.

[0012] Preferably, the autofluorescence background inhibitor is an aqueous ethanol solution containing Sudan Black B, wherein the mass fraction of Sudan Black B is 0.1-0.5% and the volume fraction of ethanol is 65-75%.

[0013] Secondly, the present invention provides a non-diagnostic detection method for a multiplex fluorescent staining kit for tissue samples, specifically comprising the following steps: S1. Sample pretreatment: Dewax and hydrate the tissue sections, add an autofluorescence background inhibitor, incubate, wash, and obtain pretreated tissue sections. S2. Single-round staining and imaging: The pretreated tissue sections are incubated with a primary antibody against the first target and washed; then incubated with an HRP-labeled secondary antibody polymer and washed; then incubated with a signal amplification deposition solution and washed; then incubated with a fluorescent labeling reagent for copper-free click chemical coupling and washed; then incubated with tissue-protective imaging medium for fluorescence imaging of the current round and saving the monochrome image data, and washed to obtain the tissue sections to be eluted; S3. Antibody elution: Place the tissue section to be eluted in antibody dissociation buffer, apply a DC electric field for elution, perform preliminary washing, immerse in neutralization buffer, wash again, and obtain regenerated sections; S4. Subsequent cycles of cyclic staining: Starting with the regenerated section, repeat steps S2 to S3, using primary antibodies against subsequent targets and different fluorescent labeling reagents until all target detections are completed. S5. Image Fusion: Spatial registration and channel overlay of the monochrome images saved in steps S2 and S4 are performed to obtain a multicolor fused image.

[0014] Preferably, the working concentration of the HRP-labeled secondary antibody polymer in step S2 is 1-5 μg / mL.

[0015] Preferably, in step S2, each wash uses PBS buffer containing 0.05-0.1 wt% Tween-20, and the washes are performed 3 times, each wash lasting 3-5 minutes.

[0016] Preferably, in step S3, the DC electric field strength is 5-15V / cm, the elution temperature is 2-8℃, and the elution time is 10-30min.

[0017] Preferably, the neutralization buffer in step S3 is a 40-60mM Tris-HCl buffer containing 140-160mM sodium chloride, with a pH of 7.4-8.0 and an immersion time of 2-5 minutes.

[0018] Preferably, the DC electric field applied in step S3 is configured to cause positively charged antibody molecules to migrate directionally away from the tissue slice and toward the negative electrode; the electrode is isolated from the area where the tissue slice is located by a porous isolation membrane to prevent electrolytic bubbles from contacting the tissue slice; the porous isolation membrane is selected from one of dialysis membrane, cellulose ester membrane, and polycarbonate membrane.

[0019] Preferably, the non-diagnostic detection method of the multiplex fluorescent staining kit for tissue samples can support 6-12 cycles of staining, with intact tissue morphology and no significant signal attenuation.

[0020] Thirdly, the present invention provides the application of a multiplex fluorescent staining kit for tissue samples in the preparation of reagents for non-diagnostic purposes such as spatial analysis of the tumor microenvironment, monitoring of transplant immune rejection, or pathological assessment of neurodegenerative diseases.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention achieves mild and efficient antibody elution, maximizing the protection of tissue integrity and antigen activity. Addressing the problems of existing technologies that often employ high-temperature microwave retrieval or strong reducing agent elution, leading to tissue morphology collapse, irreversible denaturation of antigenic epitopes, and slide detachment, this invention utilizes a salping mechanism of "low-pH buffer solution and directional electric field synergy." A low-pH glycine buffer solution reversibly disrupts the non-covalent bonds between antigen and antibody, and a specific directional DC electric field actively "pulls" the dissociated positively charged antibody away from the tissue surface, rather than relying on passive diffusion. This combination allows the elution process to be performed at low temperatures, eliminating the risks of thermal damage and chemical denaturation. This method supports 6-12 rounds of continuous cyclic staining with intact tissue morphology and no significant attenuation of antigen binding capacity in subsequent rounds, making it suitable for precious clinical puncture samples and tissue microarrays.

[0022] (2) This invention overcomes the contradiction between "penetration" and "sensitivity" in signal amplification, and improves the detection capability of low-abundance targets. Although traditional TSA technology has good penetration, its single-point signal gain is limited, while pre-polymerized macromolecular probes, although having strong signals, are difficult to penetrate into the deep layers of dense tissues due to large steric hindrance. This invention adopts a two-step cascade amplification strategy of "small molecule deposition + post-click chemistry labeling": First, cyclooctyne tyrosine molecules are deposited in situ under HRP catalysis to penetrate deep tissue layers; then, an azide-based fluorescent dye is introduced, and a large number of fluorescent molecules are covalently coupled to the deposition site through a copper-free click chemistry reaction. This strategy retains the excellent penetration of small molecules and achieves ultra-high signal amplification similar to dendritic macromolecules, improving the signal-to-noise ratio and enabling low-abundance targets (such as immune checkpoint proteins) to be clearly visualized.

[0023] (3) This invention solves the problem of bubble damage in electrophoretic elution, ensuring the reproducibility and stability of the experiment. Applying an electric field directly near tissue sections usually leads to water electrolysis generating bubbles, physically tearing the tissue, or causing imaging artifacts. This invention uses a porous isolation membrane to isolate the electrode reaction area from the tissue section area through a physical barrier, allowing ions to pass through to form an electric field, but completely blocking gas molecules from contacting the sections. This design eliminates the physical damage to the tissue microstructure caused by electrolysis bubbles, ensuring the smoothness of the tissue sections and the imaging quality under multiple rounds of electric field treatment.

[0024] (4) This invention establishes a precise electrochemical environment reset mechanism, ensuring the uniformity of multiple rounds of staining. To address the issue that residual acid after acidic elution may lead to inactivation of the primary antibody in the next round, this invention incorporates a neutralization buffer. Utilizing the high buffering capacity of Tris-HCl buffer, the pH of the tissue microenvironment is rapidly brought back from acidic to physiological neutral, simultaneously displacing residual high-salt components. This not only prevents the continuation of acid damage but also eliminates charge interference, ensuring that each new primary antibody can specifically bind under optimal physiological conditions, effectively avoiding background elevation or false negatives caused by pretreatment residues.

[0025] (5) This invention provides a highly orthogonal and stable labeling system, eliminating crosstalk between rounds. The copper-free click chemistry (cyclooctyne-azide) used has extremely high bioorthogonality, does not react with endogenous groups in tissues, and the formed triazole ring covalent bond is extremely stable under acidic elution conditions. This ensures that the fluorescence signal from the previous round does not fall off or quench after multiple strong acid and electric field treatments, while the reagents in the new round will not cross-react with non-target sites. Combining the strategy of independent imaging in each round with registration and fusion of the final image solves the common problems of spectral crosstalk and antibody species limitation in traditional multiplex staining, realizing ultra-multicolor space phenotypic analysis, and providing a powerful tool for tumor microenvironment analysis, immunotherapy evaluation, and neuropathological research. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0028] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0029] PBS buffer was purchased from Solarbio, pH 7.4, 0.01M, Cat: P1020; Hematoxylin-eosin staining solution was purchased from Solarbio, Cat: G1120.

[0030] Example 1

[0031] This embodiment provides a multiplex fluorescent staining kit for tissue samples, comprising the following components: antibody dissociation buffer, signal amplification deposition solution, fluorescent labeling reagent, tissue protective imaging medium, and autofluorescence background inhibitor; The specific formulas for each component are as follows: (1) Antibody dissociation buffer: an aqueous solution containing 100 mM glycine, 150 mM sodium chloride and 0.1 wt% Tween-20, with pH adjusted to 2.5 using 100 mM hydrochloric acid.

[0032] (2) Signal amplification deposition solution: an aqueous solution containing 10 μg / mL of DBCO-Tyrosine (dibenzocyclooctyne-tyrosine), purchased from Xi'an Ruixi Biotechnology Co., Ltd.

[0033] (3) Fluorescent labeling reagents: including aqueous solutions of AF488-azide (fluorescent labeling reagent A), AF555-azide (fluorescent labeling reagent B), AF594-azide (fluorescent labeling reagent C), AF647-azide (fluorescent labeling reagent D), AF680-azide (fluorescent labeling reagent E) and AF750-azide (fluorescent labeling reagent F), all with a concentration of 2 μg / mL.

[0034] (4) Tissue-protective imaging medium: composed of 2wt% aqueous methylcellulose, 0.3wt% aqueous vitamin E phosphate and PBS buffer in a volume ratio of 1:1:8.

[0035] (5) Autofluorescence background inhibitor: An aqueous solution of ethanol containing Sudan Black B, wherein the mass fraction of Sudan Black B is 0.1-0.5% and the volume fraction of ethanol is 65-75%.

[0036] Example 2

[0037] This embodiment provides a non-diagnostic detection method for detecting the 6-color lung cancer tissue microenvironment using the kit described in Example 1. The aim is to simultaneously detect six targets—PD-L1, CD8, CK (Pan-Cytokeratin), CD68, Ki-67, and PD-1—on a single paraffin section to analyze the spatial distribution characteristics of the tumor immune microenvironment.

[0038] Specifically, the following steps are included: S1. Sample Pretreatment: Formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma tissue sections (4 μm thick) were baked in a 65°C oven for 2 hours. After removal, the sections were placed in xylene I and xylene II sequentially, each for 10 minutes, for dewaxing. Then, they were subjected to a gradient hydration with 100% (v / v), 95% (v / v), 80% (v / v), and 70% (v / v) ethanol, and finally equilibrated in distilled water for 10 minutes. The sections were removed, and 100 μL of autofluorescence background inhibitor was added. The sections were incubated at room temperature in the dark for 8 minutes. The sections were then removed and washed three times with PBS buffer containing 0.05% (v / v) Tween-20 for 3 minutes each time to obtain the pretreated tissue sections. S2, Single-roll staining and imaging (target: PD-L1): Primary antibody incubation: Add 100 μL of rabbit anti-human PD-L1 primary antibody working solution (working concentration 5 μg / mL) to the pretreated tissue sections, incubate overnight at 4°C in a humidified chamber, and then wash the sections 3 times with PBS buffer containing 0.05% (v / v) Tween-20, each time for 3 min; Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody working solution (working concentration 2 μg / mL) to the pretreated tissue sections, incubate at room temperature in the dark for 30 min, and then wash the sections 3 times with PBS buffer containing 0.05% (v / v) Tween-20, each time for 3 min; Signal deposition: Add 100 μL of signal amplification deposition solution to the pretreated tissue sections and react at room temperature in the dark for 10 min. Then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20 for 3 min each time to allow DBCO groups to be deposited in situ at the PD-L1 antigen site. Fluorescent labeling: Add 100 μL of fluorescent labeling reagent A (containing 2 μg / mL AF488-azide) to the pretreated tissue sections and incubate at room temperature in the dark for 30 min to complete the copper-free click chemical reaction; then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20, each time for 3 min; Imaging: Add 100 μL of tissue-protective imaging medium to the pretreated tissue sections, cover with a coverslip, and use a fluorescence microscope to acquire AF488 channel fluorescence images at an excitation wavelength of 495 nm / emission wavelength of 520 nm. Name and save the images as Round1.tif. Preparation for elution: Remove the coverslip and wash the slides three times with PBS buffer containing 0.05% (v / v) Tween-20 for 3 minutes each time to remove residual imaging media and obtain the tissue slides to be eluted; S3. Antibody Elution: Place the tissue sections to be eluted in the carrying area of ​​the electrophoresis elution tank, add antibody dissociation buffer pre-cooled to 4℃ to the tank, and completely immerse the tissue sections to be eluted; use a polycarbonate membrane to isolate the electrode chamber from the section carrying area, blocking electrolytic bubbles while conducting the electric field; place the negative electrode on the opposite side of the section to construct a DC electric field that drives the positively charged antibody to migrate away from the section towards the negative electrode; set the electric field strength to 10V / cm, control the temperature at 4℃ throughout the process, and continuously elute for 20min. After elution, wash the sections twice with PBS buffer containing 0.05% (v / v) Tween-20, 1min each time, and then immerse them in neutralization buffer and incubate at room temperature for 3min to neutralize the residual acidic buffer; then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20, 3min each time, to obtain regenerated sections; S4. Subsequent cycles of staining (targets: CD8, CK, CD68, Ki-67, PD-1): Starting with the regenerated section, repeat steps S2 to S3 for a total of 5 cycles. The antibody and fluorescent dye combinations used in each cycle are shown in Table 1. Table 1. Correspondence between targets and fluorescent dyes in multi-cycle staining

[0039] Note: The same "imaging-electric field elution-neutralization" procedure was performed after each staining round. The working concentration of all fluorescent dyes was 2 μg / mL, and the click chemistry incubation time was 30 min.

[0040] S5. Image Fusion: Import the six monochrome fluorescence images acquired in the first to sixth rounds into the image analysis software; using the DAPI staining image as a reference, perform rigid registration on the images from each round to correct for displacement; superimpose and fuse the registered six-channel images to obtain a six-color fluorescence fusion image.

[0041] Experimental results: This embodiment successfully demonstrates the entire process of completing 6 rounds of cyclic staining (detecting PD-L1, CD8, CK, CD68, Ki-67, and PD-1) on a single lung cancer tissue section using the kit of this invention. Experimental results show that the method is smooth, and effective fluorescence signals can be collected in each round, confirming its ability for multicolor space phenotypic analysis and high-sensitivity detection of low-abundance targets. After 6 cycles of treatment, the tissue morphology remained intact without detachment, and there was no signal loss or crosstalk from the preceding sequences.

[0042] Example 3

[0043] The purpose of this embodiment is to verify the elution efficiency of the multiplex fluorescent staining kit of the present invention, and to compare the advantages and disadvantages of the electric field-assisted elution of the present invention with the traditional elution method by setting up a control experiment.

[0044] 1. Experimental Materials (1) Tissue samples: Formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma tissue blocks were used to prepare serial sections (4 μm thick), a total of 12 sections, which were attached to acid-resistant super-adhesive glass slides. The sections were baked in an oven at 65℃ for 2 hours before use.

[0045] (2) Reagents: ① Multiplex fluorescent staining kit of the present invention; ② Two types of traditional elution reagents: one is sodium citrate buffer (10mM, pH6.0) for high temperature microwave thermal retrieval elution; the other is SDS / DTT elution buffer (2% (w / v) SDS, 100mM DTT, 50mM Tris-HCl, pH6.8) for chemical reagent elution; ③ Rabbit anti-human broad-spectrum cytokeratin (CK) primary antibody; ④ HRP-labeled goat anti-rabbit secondary antibody; ⑤ Hematoxylin-eosin staining solution.

[0046] 2. Experimental Grouping The 12 consecutive slides were randomly divided into 4 groups of 3 slides each, and each group underwent different elution treatments: (1) The elution conditions of this invention are: antibody dissociation buffer, 10V / cm electric field, 4℃, 20min, polycarbonate membrane to isolate air bubbles, same as step S3 in Example 2; (2) Control group 1: The elution conditions were antibody dissociation buffer soaking (without electric field), 4℃, 40min; the details are as follows: S3. Antibody elution: Place the tissue sections to be eluted in a regular staining jar, add antibody dissociation buffer pre-cooled to 4°C, and completely immerse the tissue sections to be eluted. Maintain the temperature at 4°C throughout the process and soak for 40 minutes. After the process, wash the sections twice with PBS buffer containing 0.05% (v / v) Tween-20, 1 minute each time, and then immerse them in neutralization buffer and incubate at room temperature for 3 minutes to neutralize the residual acidic buffer. Then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20, 3 minutes each time, to obtain regenerated sections. (3) Control group 2: The elution conditions were microwave thermal retrieval, sodium citrate buffer, 100℃, 15min; details are as follows: S3. Antibody elution: Place the tissue sections to be eluted in a heat-resistant retrieval box, add sodium citrate buffer to completely immerse them, place the retrieval box in a microwave oven, heat to boiling and maintain for 15 min, let it cool naturally to room temperature, wash the sections twice with PBS buffer containing 0.05% (v / v) Tween-20, 1 min each time, then immerse them in neutralization buffer and incubate at room temperature for 3 min, then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20, 3 min each time, to obtain regenerated sections; (4) Control group 3: Elution conditions were chemical reagent elution, SDS / DTT elution buffer, 45℃, 15min; details are as follows: S3. Antibody elution: Place the tissue sections to be eluted in a staining jar, add SDS / DTT elution buffer to completely immerse them, place the staining jar in a 45℃ constant temperature water bath shaker, and incubate for 15 min. After that, wash the sections twice with PBS buffer containing 0.05% (v / v) Tween-20, 1 min each time, and then immerse them in neutralization buffer and incubate at room temperature for 3 min to neutralize the residual acidic buffer. Then wash the sections three times with PBS buffer containing 0.05% (v / v) Tween-20, 3 min each time, to obtain regenerated sections.

[0047] 3. Experimental Procedure S1. Sample preprocessing: Same as in Example 2; S2, Single-round staining and imaging (target: CK): The only difference from step S2 in Example 2 is that the "rabbit anti-human PD-L1 primary antibody" is replaced with "rabbit anti-human broad-spectrum cytokeratin (CK) primary antibody"; S3. Antibody elution: According to the conditions set in "2. Experimental grouping", the slides of each group were eluted accordingly to obtain the eluted slides of each group. S4. Detection of residual signals after elution: Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody working solution (2 μg / mL) to each group of eluted sections, incubate at room temperature in the dark for 30 min, and wash the sections 3 times with PBS buffer containing 0.05% (v / v) Tween-20, each time for 3 min; Fluorescent labeling: Add 100 μL of AF488-azide (2 μg / mL) to each group of slices, incubate at room temperature in the dark for 30 min, and wash three times with PBS buffer containing 0.05% (v / v) Tween-20, each time for 3 min; Residual signal acquisition: 100 μL of tissue-protective imaging medium was added to each group of slices, a coverslip was placed, and AF488 channel fluorescence images were acquired under the same imaging conditions to record the residual signal intensity. S5. Histological assessment: H&E staining: After elution, hematoxylin-eosin staining was performed on the sections of each group; Morphological scoring: Tissue morphology was observed under a microscope, and two pathologists independently scored the tissue in a blinded manner (1-5 points), and the average value was taken. Scoring criteria: intact tissue structure, clear cell boundaries, no detachment (5 points); basically intact tissue structure, occasional cell swelling (4 points); partially blurred tissue structure, widened intercellular spaces (3 points); significantly damaged tissue structure, detachment in some areas (2 points); severely disintegrated tissue, large-area detachment (1 point). S6. Antigen Retention Rate Detection: Take another set of serial sections (12 slides), perform the first round of CK staining according to steps S1 to S2 and record the initial signal intensity; after elution according to the conditions set in "2. Experimental Groups", repeat step S2 for re-staining, acquire AF488 channel fluorescence images under the same imaging conditions, record the re-staining signal intensity, and the antigen retention rate = (average fluorescence intensity of re-staining signal / average fluorescence intensity of initial signal) × 100%.

[0048] 4. Experimental Results The experimental results are summarized in Table 2.

[0049] Table 2 Comparison of the effects of different elution methods

[0050] Experimental results show that the electric field-assisted elution technology of this invention exhibits excellent performance in terms of elution efficiency, tissue protection, and antigen retention. The elution efficiency of the present invention group reached 98.4%, comparable to the SDS / DTT strong reducing agent group, and higher than the group without electric field immersion and the microwave thermal repair group. Simultaneously, the tissue morphology score of the present invention group reached 4.8 points, showing no significant difference from the group without electric field immersion, but far superior to the microwave thermal repair group and the SDS / DTT group; the antigen retention rate reached 96.1%, higher than the microwave thermal repair group and the SDS / DTT group.

[0051] Example 4

[0052] The purpose of this embodiment is to verify the cyclic staining tolerance of the multiplex fluorescent staining kit of the present invention, and to evaluate the maximum number of cycles that can be supported and the signal stability.

[0053] 1. Experimental Materials (1) Tissue samples: Formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma tissue blocks were used to prepare serial sections (4 μm thick), a total of 3 sections, which were attached to acid-resistant super-adhesive glass slides. The sections were baked in an oven at 65℃ for 2 hours before use.

[0054] (2) Reagents: ① Multiplex fluorescent staining kit of the present invention; ② Rabbit anti-human broad-spectrum cytokeratin (CK) primary antibody; ③ HRP-labeled goat anti-rabbit secondary antibody; ④ Hematoxylin-eosin staining solution.

[0055] 2. Experimental Grouping Three consecutive slides were used as parallel samples. Each slide underwent 12 rounds of cyclic staining-elution, with the same target (CK) detected in each round, but using different fluorescent labeling reagents. The arrangement of fluorescent dye rounds is shown in Table 3.

[0056] Table 3. Fluorescent dye rotation arrangement

[0057] 3. Experimental Procedure S1. Sample preprocessing: Same as in Example 2; S2, Single-round staining and imaging (target: CK): The only difference from step S2 in Example 2 is that the "rabbit anti-human PD-L1 primary antibody" is replaced with "rabbit anti-human broad-spectrum cytokeratin (CK) primary antibody". S3, Antibody elution: Same as in Example 2; S4, Cyclic staining in rounds 2-12 (target is CK): Starting from the regenerated section, repeat steps S2 to S3 for a total of 11 cycles, using the fluorescent labeling reagents corresponding to Table 3 in each cycle. S5. Histological assessment: H&E staining: After elution in rounds 3, 6, 9 and 12, one slide was taken for hematoxylin-eosin staining. Morphological scoring: Tissue morphology was observed under a microscope and scored independently by two pathologists using a blind method (1-5 points), and the average value was taken; scoring criteria: same as in Example 3.

[0058] S6. Antigen binding capacity test: Calculate the average fluorescence intensity of each round of CK staining, taking the average fluorescence intensity of the first round as 100%, and calculate the relative signal intensity of each round; relative signal intensity (%) = (average fluorescence intensity of the nth round / average fluorescence intensity of the first round) × 100%.

[0059] 4. Experimental Results The experimental results of each group are summarized in Table 4.

[0060] Table 4 Results of Cyclic Stain Tolerance Test

[0061] Experimental results show that after 12 rounds of cyclic staining-elution: antigen binding capacity remained good: the CK target signal intensity remained at 82.9% of that in the first round, and the signal attenuation was gradual, indicating that the method of the present invention can effectively maintain antigen binding capacity and support multiple rounds of continuous detection; tissue morphology integrity was good: the tissue morphology score still reached 4.5 points, and H&E staining showed that the tissue structure was intact, with no detachment or obvious disintegration, proving that the design of the present invention of low-temperature electric field assisted elution combined with polycarbonate membrane to isolate air bubbles has a significant effect on tissue protection.

[0062] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A multiplex fluorescent staining kit for a tissue sample, characterized by, It includes the following components: antibody dissociation buffer, signal amplification deposition solution, fluorescent labeling reagent, tissue-protective imaging medium, and autofluorescent background inhibitor.

2. The multiplex fluorescent staining kit for tissue samples according to claim 1, characterized in that, The antibody dissociation buffer is an aqueous solution containing 50-200 mM glycine, 300-500 mM sodium chloride and 0.05-0.2 wt% Tween-20, with a pH of 2.2-3.

0.

3. The multiplex fluorescent staining kit for tissue samples according to claim 1, characterized in that, The signal amplification deposition solution is an aqueous solution containing 5-20 μg / mL of HRP catalytic substrate, wherein the HRP catalytic substrate is a compound containing both a tyrosine structure and a first click chemical reaction functional group; The functional group of the first click chemical reaction is cyclooctyne.

4. The multiplex fluorescent staining kit for tissue samples according to claim 1, characterized in that, The fluorescent labeling reagent is an aqueous solution containing 2-10 μg / mL of a fluorescent dye coupled with a second click chemistry functional group; the second click chemistry functional group is an azide group; the fluorescent dye is selected from one or more of AF488, AF555, AF594, AF647, AF680 and AF750.

5. The multiplex fluorescent staining kit for tissue samples according to claim 1, characterized in that, The tissue-protective imaging medium is composed of 1-3 wt% aqueous methylcellulose solution, 0.1-0.5 wt% aqueous vitamin E derivative solution, and PBS buffer in a volume ratio of 1:1:6-10.

6. The multiplex fluorescent staining kit for tissue samples according to claim 1, characterized in that, The autofluorescence background inhibitor is an aqueous ethanol solution containing Sudan Black B, wherein the mass fraction of Sudan Black B is 0.1-0.5% and the volume fraction of ethanol is 65-75%.

7. A non-diagnostic detection method for a multiplex fluorescent staining kit for tissue samples according to any one of claims 1-6, characterized in that, Specifically, the following steps are included: S1. Sample pretreatment: Dewax and hydrate the tissue sections, add an autofluorescence background inhibitor, incubate, wash, and obtain pretreated tissue sections. S2, Single-round staining and imaging: The pretreated tissue sections are incubated with a primary antibody against the first target and then washed; Then incubate with HRP-labeled secondary antibody polymer, followed by washing; Add signal amplification deposition solution for incubation, then wash; add fluorescent labeling reagent for incubation, perform copper-free click chemical reaction coupling, then wash; Then add tissue-protective imaging medium, perform fluorescence imaging for the current round and save monochrome image data, clean, and obtain tissue sections to be eluted; S3. Antibody elution: Place the tissue section to be eluted in antibody dissociation buffer, apply a DC electric field for elution, perform preliminary washing, immerse in neutralization buffer, wash again, and obtain regenerated sections; S4. Subsequent cycles of cyclic staining: Starting with the regenerated section, repeat steps S2 to S3, using primary antibodies against subsequent targets and different fluorescent labeling reagents until all target detections are completed. S5. Image Fusion: Spatial registration and channel overlay of the monochrome images saved in steps S2 and S4 are performed to obtain a multicolor fused image.

8. The non-diagnostic detection method of the multiplex fluorescent staining kit for tissue samples according to claim 7, characterized in that, In step S3, the DC electric field strength is 5-15V / cm, the elution temperature is 2-8℃, and the elution time is 10-30min.

9. The non-diagnostic detection method of the multiplex fluorescent staining kit for tissue samples according to claim 7, characterized in that, The neutralization buffer in step S3 is a 40-60mM Tris-HCl buffer containing 140-160mM sodium chloride, with a pH of 7.4-8.0, and an immersion time of 2-5 minutes.

10. The use of the multiplex fluorescent staining kit for tissue samples according to any one of claims 1-6 in the preparation of reagents for non-diagnostic purposes such as spatial analysis of the tumor microenvironment, monitoring of transplant immune rejection, or pathological assessment of neurodegenerative diseases.

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