A multiplex analysis method for target proteins in tissue sections based on reversible DNA self-assembly

Abstract

The invention discloses a tissue slice target protein multiple analysis method based on reversible DNA self-assembly, comprising step one, design of a DNA self-assembly system, step two, synthesis of an antibody Ab-T, step three, pretreatment and imaging of the target protein of the tissue slice, and step four, image processing of imaging analysis; the invention combines DNA self-assembly system design, click chemistry reaction, immune recognition, reversible DNA self-assembly and tissue fluorescence imaging technology, outputs multiple target proteins on the tissue slice as multiple fluorescence signals, realizes multiple analysis of the target proteins on the tissue slice, reduces time cost and demand for tissue samples without reducing imaging accuracy; and has important significance for basic scientific research, clinical research and application such as spatial localization relationship analysis of multiple target proteins of tissue samples, especially precious tissue samples, cell typing analysis, tissue spatial distribution analysis of cells, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of high-throughput detection, and in particular to a tissue section target protein multiple analysis method based on reversible DNA self-assembly. Background Art

[0002] With the vigorous development of molecular pathology technology, more and more disease-related molecular changes have been discovered and gradually applied in clinical practice. Since the 21st century, molecular pathology diagnosis has gradually been recognized and valued by pathologists in my country, and has gradually been carried out in hospital pathology departments. Especially in the study of tumors, molecular pathology diagnosis has made great progress. Tissue molecular pathology analysis is the detection of tumor pathological tissue sections. Its significance lies in 1) diagnosis and differential diagnosis of malignant tumors; 2) determination of the primary site of metastatic malignant tumors; 3) further pathological classification of certain types of tumors; 4) indispensable for the diagnosis and treatment of soft tissue tumors; 5) discovery of micrometastases, which helps to determine clinical treatment plans and surgical scope; 6) providing treatment options for clinical practice. Therefore, tissue molecular pathology analysis has an urgent need in both scientific research and clinical practice. At the same time, spatial and high-resolution molecular pathology analysis of cells in pathological tissues will also be a frontier field in future scientific research and clinical practice. However, for tissue pathology, the occurrence and development of the disease is not caused by a single factor, so the diagnosis of a single marker is limited.

[0003] Currently, fluorescence imaging technology can be used for multiplex analysis of tissue pathology. Through fluorescence imaging, the fluorescent image in the visual dimension is directly linked to the molecular phenotype of the marker. However, the spectral overlap problem will limit the detection throughput. Only one marker can be labeled in each round, the analysis time is long, and high-throughput analysis cannot be performed. Therefore, the present invention proposes a multiplex analysis method of tissue section target protein based on reversible DNA self-assembly to solve the problems existing in the prior art. Summary of the invention

[0004] In view of the above problems, the purpose of the present invention is to propose a multiplex analysis method for target proteins in tissue sections based on reversible DNA self-assembly, which combines click chemistry reaction, immune recognition, reversible DNA self-assembly and fluorescence imaging technology to achieve multiplex analysis of pathological tissue, reduce time cost without reducing imaging accuracy, and output multiple marker proteins on pathological tissue sections as multiple fluorescent signals, thereby performing high-throughput analysis.

[0005] To achieve the purpose of the present invention, the present invention is implemented by the following technical scheme: a method for multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly, including the application of the method in the analysis of paraffin tissue sections, frozen tissue sections and tissue-like samples fixed on an imaging interface, comprising the following steps:

[0006] Step 1: Design of a DNA self-assembly system, according to the principle that both the T sequence used for DNA self-assembly and the D sequence used for DNA deassembly do not contain a G base;

[0007] Step 2: Synthesis of antibody Ab-T: synthesizing multiple groups of T chains that can specifically initiate self-assembly of different DNAs, and then labeling them on specific first antibodies Ab based on click chemistry to obtain multiple different antibodies Ab-T;

[0008] Step 3: Pretreatment and imaging of target proteins in tissue sections. First, the tissue sections are subjected to antigen activation treatment. The specificity of antigen-antibody immune recognition is used to perform specific Ab-T labeling on different protein targets to be analyzed. The labeled tissue sections are then subjected to cyclic DNA self-assembly imaging and disassembly imaging.

[0009] Step 4: Image processing for imaging analysis: complete images of different target proteins marked with different colors are obtained through image alignment, and image processing programs are used to analyze the subcellular spatial localization, relative position relationship, cell molecular typing, cell-to-cell interaction relationship, and cell-to-cell spatial localization relationship of each protein;

[0010] The further improvement lies in that the specific method in the step 2 is to firstly cross-link the high concentration of m×n first antibodies Ab with the high concentration of NHS-PEG4-N3 in a 4°C magnetic stirrer for 2 hours to obtain Ab-N3; then, Ab-N3 is mixed with T-DBCO, reacted in a 4°C magnetic stirrer for 1 hour, and then transferred to a 4°C environment for further reaction for 24 hours to obtain m×n antibody-primer complex Ab-T.

[0011] Further improvements are: the speed of the magnetic stirrer is controlled at 1000-1200rpm, the concentration of the first antibody Ab used is ≥1mg / mL, the concentration of NHS-PEG4-N3 used is 20-50 times the concentration of the first antibody Ab, and DMSO is used for dissolution, the reaction system of the two is 40-100μL, wherein the content of DMSO in the reaction system is less than 5%; T-DBCO is a trigger chain T modified by DBCO, and the molar concentration ratio of T-DBCO to Ab-N3 is 3:1.

[0012] A further improvement is that in step three, the tissue sections are first subjected to antigen repair, specifically, the tissue paraffin sections are first immersed in antigen repair solution, heated to boiling in a 650W microwave oven at 60% power for 8 minutes, then stopped and allowed to stand for 8 minutes, then reheated in a 650W microwave oven at 40% power for 8 minutes, and finally cooled to room temperature.

[0013] A further improvement is that when performing specific Ab-T labeling in step 3, non-specific sites are first blocked with immunostaining blocking solution for 1 hour, and then a variety of different antibodies Ab-T are added to the blocked tissue sections for incubation overnight.

[0014] A further improvement is that in step three, m protein targets are imaged in each cycle, and imaging is performed for n cycles to achieve analysis of m×n protein targets, where m≤6.

[0015] A further improvement is that: the step three specific Ab-T labeling specifically includes

[0016] S1. Marker labeling: using the characteristics of antigen-antibody immune recognition, multiple different antibodies Ab-T are added to the tissue section at the same time to achieve specific binding labeling of different protein markers to be analyzed;

[0017] S2, imaging analysis processing, imaging processing of the labeled tissue slices, in the first imaging cycle, there are m kinds of protein markers labeled with antibody Ab-T, which orthogonally initiate m groups of independent DNA self-assembly, and are labeled with m different fluorescent groups to complete in-situ amplified imaging analysis of m kinds of protein markers, where m≤5;

[0018] S3, self-assembly product disassembly treatment, after the first imaging cycle imaging analysis is completed, the disassembly chain D corresponding to each group of DNA self-assembly is added to undergo a toehold-mediated chain displacement reaction with the self-assembly product to complete the disassembly, and the fluorescence signal disappears.

[0019] A further improvement is that: in S1, the final concentration of A1 and A2 modified with fluorescent groups corresponding to multiple groups of different antibodies Ab-T are added to S3 on tissue sections to be 500nM, the volume is 100-200μL, and it is left to react at 37°C for 1-2h.

[0020] A tissue section target protein multiplex analysis method based on reversible DNA self-assembly is applied to the cyclic imaging of a small number of markers with a single marker per cycle or multiple markers per cycle and the multiple imaging of a large number of markers on whole tissue samples (including pathological tissue, normal tissue and tissue-like samples fixed on an imaging interface).

[0021] A tissue section target protein multiplex analysis method based on reversible DNA self-assembly uses a single marker per cycle or multiple markers per cycle on the whole tissue sample with other fluorescent combinations for cycle imaging of a small number of markers and multiple imaging of a large number of markers.

[0022] The beneficial effects of the present invention are as follows: the present invention combines the design of a DNA self-assembly system, click chemistry, immune recognition, reversible DNA self-assembly and fluorescence imaging technology to achieve multiplexed analysis of target proteins on tissue sections.

[0023] The method of the present invention does not involve species-specific primary and secondary antibodies during the analysis process, thus greatly increasing the analysis throughput of tissue protein targets; the introduction of the reversible DNA self-assembly technology of the disassembly chain D makes the disassembly process shorter than the thermal repair elution method, and the bound primary antibody is more stable; the replacement of the fluorescent secondary antibody with the reversible DNA self-assembly technology with signal amplification can make it easier to detect tissue protein targets with lower abundance; through multiple rounds of imaging analysis, the imaging efficiency of the tissue protein targets to be imaged will be greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of the method of Example 1 of the present invention.

[0025] Figure 2 This is a schematic diagram of the reversible DNA self-assembly reaction of Example 1 of the present invention.

[0026] Figure 3 This is an imaging diagram of the performance retention of the antibody before and after modification in Example 1 of the present invention.

[0027] Figure 4 This is the simultaneous analysis and imaging of three target proteins in colorectal cancer tissue in Example 1 of the present invention.

[0028] Figure 5 This is an imaging diagram of the target protein circulation analysis in colorectal cancer tissue according to Example 1 of the present invention.

[0029] Figure 6 This is a graph of multiple rounds of multiple analysis of target proteins in colorectal cancer tissue in Example 2 of the present invention. DETAILED DESCRIPTION

[0030] In order to deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with examples. The examples are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0031] Example 1

[0032] according to Figure 1-Figure 5As shown, this embodiment provides a method for multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly. The reversible DNA self-assembly mentioned is composed of an antibody Ab-T modified with an initiator chain, two hairpin DNA chains A1 and A2 with a stem-loop structure and a foothold, and a disassembly chain D. After Ab-T binds specifically to the tissue through antigen-antibody immune recognition and is washed, Ab-T marked on the pathological tissue can induce A1 and A2 to undergo a DNA self-assembly amplification reaction, and A1 and A2 can be modified with any fluorescent group to achieve fluorescent imaging of tissue pathological markers. After the disassembly chain D is introduced, D will undergo a foothold-mediated chain displacement reaction with the assembly product, and the self-assembled product will disintegrate, and the fluorescent signal will disappear.

[0033] The specific steps include:

[0034] Step 1: Design of the DNA self-assembly system. All T sequences used for DNA self-assembly and D sequences used for DNA deassembly do not contain G bases to avoid interference within and between sequences.

[0035] Step 2, synthesis of antibody Ab-T, synthesizing multiple groups of T chains that can specifically initiate different DNA self-assemblies, and then labeling them on specific first antibodies Ab based on click chemistry to obtain a variety of different antibodies Ab-T; the specific method is to firstly stir 1 mg / mL of m×n first antibodies Ab with 0.2 mM NHS-PEG4-N3 in a 4°C magnetic stirrer at a speed of 1000-1200 rpm for 2 h for cross-linking reaction, so that the amino group of the antibody is converted into an azide group to obtain Ab-N3; then, different Ab-N3s are mixed with corresponding T-DBCO at a concentration ratio of 1:3, and the reaction is continued for 24 h in a 50 μL system in a 4°C magnetic stirrer at a speed of 1000-1200 rpm, and the product is subjected to 5 The mixture was centrifuged in a 0kD ultrafiltration centrifuge tube and washed 5 times with 1×PBS to obtain the product Ab-T, which was redissolved to 1 mg / mL (6.67 μM) to obtain m×n antibody-primer complexes Ab-T. The antibodies were diluted in advance according to the manufacturer's recommended concentration before use; the concentration of the first antibody Ab used was ≥1 mg / mL. If the concentration of the first antibody Ab did not meet the required requirements, an ultrafiltration tube was required to concentrate and purify the antibody. The concentration of NHS-PEG4-N3 used was 20-50 times that of the first antibody Ab, and DMSO was used to dissolve it. The reaction system of the two was 40-100 μL, and the content of DMSO in the reaction system was less than 5%; T-DBCO was a trigger chain T modified with DBCO, and the molar concentration ratio of T-DBCO to Ab-N3 was 3:1.

[0036] Step 3: Pretreatment and imaging of target proteins in tissue sections. First, the tissue sections are subjected to antigen activation treatment. The specificity of antigen-antibody immune recognition is used to perform specific Ab-T labeling on different protein targets to be analyzed. The labeled tissue sections are then subjected to cyclic DNA self-assembly imaging and disassembly imaging.

[0037] The specific steps include:

[0038] S1. Marker labeling: using the characteristics of antigen-antibody immune recognition, multiple different antibodies Ab-T are added to the tissue section at the same time, and different protein targets to be analyzed are specifically labeled at the same time, that is, one protein target specifically corresponds to one antibody Ab-T, and each protein target corresponds to a different T;

[0039] The tissue sections are first subjected to antigen repair, specifically, the prepared colorectal cancer paraffin sections are placed in a 65°C oven for 2 hours in advance to melt the paraffin. The paraffin sections are placed in xylene for 30 minutes - xylene for 30 minutes - xylene for 30 minutes - anhydrous ethanol for 5 minutes - anhydrous ethanol for 5 minutes - 95% ethanol for 5 minutes - 75% ethanol for 5 minutes - distilled water for 5 minutes - distilled water for 5 minutes to rinse the sections to dewax and rehydrate them. The next step is to perform antigen repair, place the sections in EDTA antigen repair solution, first heat them in a microwave oven on medium heat for 8 minutes to boil, turn off the heat to keep warm for 8 minutes, heat on medium-low heat for 7 minutes, cool naturally, and then wash them 3 times with PBS buffer, 5 minutes each time;

[0040] The immunohistochemistry pen draws a circle around the tissue. When marking, first use 100 μL of immunostaining blocking solution to cover all tissues and block non-specific sites for 1 hour to reduce background signals. Then add 100 μL of various antibodies Ab-T or Ab to the blocked tissue sections, place them in a wet box with water, and incubate them at 4°C overnight. Take out the sections the next day and wash them 5 times with PBST, 5 minutes each time.

[0041] After the Ab-T or Ab was washed, it was incubated again with fluorescent secondary antibody for 1 hour, and the Ab activity was analyzed by imaging. The specific results are shown in the attached manual. Figure 3 shown.

[0042] S2. Imaging analysis and processing: imaging processing is performed on the labeled tissue slices. In the first imaging cycle, there are m protein targets labeled with antibody Ab-T, which orthogonally initiate m groups of independent DNA self-assemblies and are labeled with m different fluorescent groups to complete in situ amplified imaging analysis of the m protein targets, where m≤6. Using a confocal microscope, m different fluorescence of the m protein targets in situ on the tissue slices can be observed.

[0043] The specific method is to add 100 μL of a solution containing 500 nM A1 and 500 nM fluorescently labeled A2, incubate at 37°C in the dark for 60 min, and then wash with PBS three times, 5 min each time. Remove PBS, add DAPI, and in the dark for 10 min, then wash with PBS three times, 5 min each time. Cover the slices with glass slides and photograph them under a confocal microscope. The specific results are as attached in the manual. Figure 4 shown.

[0044] S3, disassembly of self-assembly products. After the samples are photographed, the coverslip is removed, and the disassembly chains D corresponding to each group of DNA self-assembly are added to undergo a toehold-mediated chain displacement reaction with the self-assembly products. After incubation at 37°C in the dark for 60 minutes, the disassembly is completed and the fluorescence signal disappears. The slices are covered with coverslips and photographed again under a confocal microscope, with the same shooting parameters before and after. Specific results are shown in the attached manual. Figure 5 As shown, it includes self-assembly and disassembly.

[0045] The final concentration of A1 and A2 modified with fluorescent groups corresponding to the multiple groups of different antibodies Ab-T used on tissue sections in S2 and the multiple groups of disassembly chains D added in S3 is 100-500nM, the volume is 100-200μL, and they are left to react at 37°C for 1-2h; the trigger chain T, assembly chains A1, A2 and disassembly chain D need to react strictly orthogonally, and each group of trigger chains T corresponds to its own assembly chains A1, A2 and disassembly chain D.

[0046] After the self-assembly product is disassembled, another m protein targets to be analyzed labeled with antibody Ab-T are imaged and analyzed in a second imaging cycle through DNA self-assembly. After the imaging analysis is completed, the disassembly chains D corresponding to each group of DNA self-assembly in step 5 are added for disassembly again. After n cycles, the m×n target proteins are imaged and analyzed through m×n groups of DNA self-assembly sequences.

[0047] Due to the overlap of fluorescence spectra, only m (m≤6) fluorescent groups can be selected. Therefore, at most m target proteins can be imaged in each round. After n cycles, m×n target proteins can be successfully imaged to achieve multiplex analysis of the whole tissue, where n represents the number of cycles of imaging analysis.

[0048] Step 4: Image processing for imaging analysis: Computer image alignment is used to obtain complete images of different target proteins marked with different colors, and image processing programs are used to analyze the subcellular spatial localization, relative position relationship, cell molecular typing, cell-to-cell interaction relationship, and cell-to-cell spatial localization relationship of each protein.

[0049] Example 2

[0050] according to Figure 6As shown, this embodiment provides a multiplex analysis of colorectal cancer tissue pathological protein markers based on reversible DNA self-assembly, as follows:

[0051] The 9 1mg / mL first antibodies Ab were cross-linked with 0.2mM NHS-PEG4-N3 in a magnetic stirrer at 4℃ for 2h, 1000rpm, to convert the amino group of the antibody into an azide group. Ab-N3 was mixed with 9 different sequence T-DBCO at a concentration ratio of 1:3, and 200μL system was magnetically stirred at 1000rpm at 4℃ for 24h. The product was centrifuged through a 50kD ultrafiltration centrifuge tube and washed 5 times with 1×PBS to obtain 9 products Ab-T, which were redissolved to 6.67μM and diluted 100-1000 times for standby use. The specific dilution multiples were operated as described in the antibody manual. The prepared colorectal cancer paraffin sections were placed in a 65℃ oven for 2h in advance to melt the paraffin. Place the paraffin sections in xylene for 30 minutes-xylene for 30 minutes-xylene for 30 minutes-anhydrous ethanol for 5 minutes-anhydrous ethanol for 5 minutes-95% ethanol for 5 minutes-75% ethanol for 5 minutes-distilled water for 5 minutes-distilled water for 5 minutes to dewax and rehydrate the sections. The next step is antigen repair. Place the sections in EDTA antigen repair solution, first heat them in a microwave oven for 8 minutes on medium heat to boil, turn off the heat for 8 minutes to keep warm, medium and low heat for 7 minutes, cool naturally, and then wash them 3 times with PBS buffer, each for 5 minutes. Slightly dry the sections, draw circles around the tissue with an immunohistochemistry pen, then add 100μL of immunostaining blocking solution, cover all tissues, and block them on a shaker at room temperature for 60 minutes. Remove the immunostaining blocking solution and place it on the experimental paper, add 100μL of Ab-T after 1-9 different dilutions, place it in a wet box with water, and incubate it overnight at 4℃. The next day, the slices were removed and washed with PBST for 5 times, 5 min each time, and 100 μL of 500 nM A1 and A2 corresponding to the T chains of the G1-G3 groups were added. After incubation at 37°C in the dark for 60 min, the slices were washed with PBS for 3 times, 5 min each time, and the PBS was removed. The slices were covered with glass slides and photographed under a confocal microscope. After the shooting was completed, the coverslip was removed, and the disassembly chains D corresponding to the three groups of DNA self-assembly imaged this time were added. After incubation at 37°C in the dark for 60 min, the slices were washed with PBS for 3 times, 5 min each time. The slices were covered with coverslips and photographed again under a confocal microscope. After the shooting was completed, the coverslip was removed, and A1 and A2 corresponding to the T chains of the G4-G6 groups were added. The reaction system contained 100 μL of 500 nM A1 and A2 corresponding to the three groups of T chains. After incubation at 37°C in the dark for 60 min, the slices were washed with PBS for 3 times, 5 min each time. The slices were covered with glass slides and photographed under a confocal microscope. After the shooting is completed, remove the coverslip, add the disassembled chains D corresponding to the three groups of DNA self-assemblies imaged this time, incubate at 37℃ in the dark for 60min, and wash with PBS 3 times, 5min each time. Cover the slice with a coverslip and shoot again under a confocal microscope. Finally, repeat the same operation for 7-9 groups, and shoot 5 times in total. The shooting parameters of the same fluorescence before and after all images are set the same.The groups of antibodies and reversible DNA corresponding to each antigen are shown in the following table, and the specific results are attached to the instructions. Figure 6 shown.

[0052] Table of antibodies and reversible DNA groups corresponding to each target protein

[0053] Each target protein Trigger Chain Unmodified A1 Fluorescent modified A2 Disassembly chain CK20 T1 G1-A1 G1-A2-FAM D1 CD34 T2 G2-A1 G2-A2-Cy3 D2 AE1 / AE3 T3 G3-A1 G3-A2-Cy5 D3 D2-40 T4 G4-A1 G4-A2-FAM D4 CA19-9 T5 G5-A1 G5-A2-Cy3 D5 BRAF T6 G6-A1 G6-A2-Cy5 D6 C-erbB-2 T7 G7-A1 G7-A2-FAM D7 CK7 T8 G8-A1 G8-A2-Cy3 D8 Villin T9 G9-A1 G9-A2-Cy5 D9

[0054] From the above examples, it can be seen that in the application of colorectal cancer tissue sections, by selecting 3 types of fluorescence and 3 cycles, the analysis of target proteins in 9 tissue samples was initially achieved. Through expansion, it can be applied to paraffin tissue sections, frozen tissue sections, and tissue-like samples fixed on the imaging interface. This is of great significance for the spatial localization relationship analysis of multiple target proteins in basic scientific research, clinical research and application, cell typing analysis, and tissue spatial distribution analysis of cells.

[0055] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly, characterized in that: The method is applied in the analysis of paraffin tissue sections, frozen tissue sections and tissue-like samples fixed on an imaging interface, comprising the following steps: Step 1: Design of a DNA self-assembly system, according to the principle that both the T sequence used for DNA self-assembly and the D sequence used for DNA deassembly do not contain a G base; Step 2: Synthesis of antibody Ab-T: synthesizing multiple groups of T chains that can specifically initiate self-assembly of different DNAs, and then labeling them on the first antibody Ab based on click chemistry to obtain multiple different antibodies Ab-T; The specific method is to firstly mix high concentration m×n first antibodies Ab with high concentration The cross-linking reaction was carried out in a magnetic stirrer at 4°C for 2 h to obtain Ab- ; Ab- Mix with T-DBCO, react in a 4 ℃ magnetic stirrer for 1 hour, then transfer to a 4 ℃ environment and continue to react for 24 hours to obtain m×n antibody-primer complex Ab-T; The speed of the magnetic stirrer was controlled at 1000-1200 rpm, the concentration of the first antibody Ab used was ≥1 mg / mL, and the The concentration of the first antibody Ab is 20-50 times that of the first antibody Ab, and it is dissolved in DMSO. The reaction system of the two is 40-100 μL, wherein the content of DMSO in the reaction system is less than 5%; T-DBCO is a trigger chain T modified by DBCO, and the molar concentration ratio of T-DBCO to Ab- is 3:1; Step 3: Pretreatment and imaging of target proteins in tissue sections. First, the tissue sections are subjected to antigen activation treatment. The specificity of antigen-antibody immune recognition is used to perform specific Ab-T labeling on different protein targets to be analyzed. The labeled tissue sections are then subjected to cyclic DNA self-assembly imaging and disassembly imaging. Step 4: Image processing for imaging analysis: complete images of different target proteins marked with different colors are obtained through image alignment, and image processing programs are used to analyze the subcellular spatial localization, relative position relationship, cell molecular typing, cell-to-cell interaction relationship and cell-to-cell spatial localization relationship of each protein.

2. The method of multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly according to claim 1, characterized in that: In the step 3, the tissue sections are first subjected to antigen repair, specifically, the tissue paraffin sections are first immersed in the antigen repair solution, heated in a 650 W microwave oven at 60% power for 8 min until boiling, then stopped and allowed to stand for 8 min, then reheated in a 650 W microwave oven at 40% power for 8 min, and finally cooled to room temperature.

3. The method for multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly according to claim 1, characterized in that: When performing specific Ab-T labeling in step 3, the non-specific sites are first blocked with an immunostaining blocking solution for 1 h, and then a variety of different antibodies Ab-T are added to the blocked tissue sections for incubation overnight.

4. The method of multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly according to claim 1, characterized in that: In the step three, m protein targets are imaged in each cycle, and n cycles are imaged to achieve analysis of m×n protein targets, where m≤6.

5. The method of multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly according to claim 1, characterized in that: The specific Ab-T labeling in step three specifically includes S1. Marker labeling: using the characteristics of antigen-antibody immune recognition, multiple different antibodies Ab-T are added to the tissue section at the same time to achieve specific binding labeling of different protein markers to be analyzed; S2, imaging analysis processing, imaging processing of the labeled tissue slices, in the first imaging cycle, there are m kinds of protein markers labeled with antibody Ab-T, which orthogonally initiate m groups of independent DNA self-assembly, and are labeled with m different fluorescent groups to complete in-situ amplified imaging analysis of m kinds of protein markers, where m≤5; S3, self-assembly product disassembly treatment, after the first imaging cycle imaging analysis is completed, the disassembly chain D corresponding to each group of DNA self-assembly is added to undergo a toehold-mediated chain displacement reaction with the self-assembly product to complete the disassembly, and the fluorescence signal disappears.

6. The method of multiple analysis of target proteins in tissue sections based on reversible DNA self-assembly according to claim 5, characterized in that: In the S1, the fluorescent group-modified A1 and A2 corresponding to multiple groups of different antibodies Ab-T are used on tissue sections. The final concentration after adding the disassembled chain D to S3 is 500 nM, the volume is 100-200 μL, and it is allowed to react at 37°C for 1-2 h.

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