Tissue sealing reagent, multi-index RNA-protein co-dyeing method and application

Abstract

The invention discloses a tissue sealing reagent, a multi-index RNA-protein co-dyeing method and application, and belongs to the technical field of molecular biology. The tissue sealing reagent comprises the following components: ethylene carbonate, dextran sulfate, sodium chloride, citric acid, trisodium citrate dihydrate and herring sperm DNA (deoxyribonucleic acid). The invention further discloses a multi-index RNA-protein co-dyeing method, multi-index co-dyeing of RNA-protein on the same tissue slice can be achieved, multiple RNA in-situ hybridization strategies are compatible, and an effective solution is provided for the non-specific dyeing problem occurring in the existing hybridization strategies. By optimizing sample treatment, a co-staining method and a signal enhancement strategy, high-resolution spatial localization of gene expression on the tissue slice is achieved, the method is particularly suitable for research of cell distribution and gene expression modes of complex tissues, and important technical support can be provided for research of disease mechanisms, development of targeted therapy and precision medicine.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology, and in particular to a tissue sealing reagent, a multi-index RNA-protein co-staining method and applications. Background Art

[0002] The study of the tumor microenvironment is a key topic in tumor biology. Traditional DNA, RNA sequencing and flow cytometry provide important molecular information, but these technologies lack spatial location information. In recent years, the rapid development of spatial omics technology has enabled the combination of molecular data and tissue structure, providing a new perspective for tumor biology research. However, since nucleic acid and protein research involves different detection technologies, the research of the two is usually carried out separately. Therefore, in practical applications, it is usually necessary to conduct nucleic acid omics and proteomics research separately. There is a lack of an efficient means to combine the two for comprehensive analysis, which restricts the comprehensive understanding of the tumor microenvironment.

[0003] Tyramide signal amplification (TSA) is an enzymatic detection method that uses horseradish peroxidase (HRP) to perform high-density in situ labeling of target proteins or nucleic acids. ® , traditional fluorescent dyes and colorimetric detection systems. By using TSA technology, low-abundance targets that cannot be detected by traditional methods can be detected. Tyramide-based signal amplification technology can provide unprecedented sensitivity without sacrificing resolution. Only a simple incubation step is required to process the sample to obtain the multi-color labeling signal of the sample; at the same time, this technology can be used in combination with other signal amplification systems or traditional labeling methods for co-localization related studies. However, the existing TSA technology is limited in its application by the problems of RNA and protein being unable to be compatible with co-staining on the same tissue section, insufficient multi-index co-staining ability, and non-specific staining interference. Therefore, it is urgent to develop a TSA staining technology that can co-stain RNA and protein. Summary of the invention

[0004] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a tissue blocking reagent, a multi-index RNA-protein co-staining method and application, so as to solve the problems in the existing TSA staining technology that RNA and protein cannot be compatible with co-staining on the same tissue section, the multi-index co-staining ability is insufficient, and non-specific staining interference.

[0005] The technical solution of the present invention to solve the above technical problems is as follows: a tissue sealing reagent is provided, comprising the following components with final concentrations: 100-200 mg / mL ethylene carbonate, 150-250 mg / mL dextran sulfate, 30-40 mg / mL sodium chloride, 2-5 mg / mL citric acid, 25-30 mg / mL trisodium citrate dihydrate and 1-5 mg / mL herring sperm DNA.

[0006] Further, the tissue blocking reagent includes the following components at final concentrations: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate, and 1 mg / mL herring sperm DNA.

[0007] The present invention provides an application of the above-mentioned tissue sealing reagent in a multi-index RNA-protein co-staining method.

[0008] The present invention provides a multi-index RNA-protein co-staining method, comprising the following steps: (1) Sample permeabilization treatment: add a probe penetrating reagent to the tissue section until the entire tissue sample is covered, and treat for 10-20 minutes; wherein the probe penetrating reagent is 1wt% Tritonx-100 dissolved in 1×PBS; (2) Tissue blocking treatment: add the above-mentioned tissue blocking reagent to the tissue section of step (1) until the entire tissue sample is covered, treat for 40 min to 60 min, and then wash with PBSTR solution and 2×SSC in sequence; (3) Perform RNA in situ hybridization; perform in situ hybridization using RNAscope technology; (4) Perform protein immunohistochemical staining.

[0009] Furthermore, the tissue sample in step (1) is a human or animal tissue sample.

[0010] Furthermore, the PBSTR solution in step (2) includes the following components: 1×PBS, 0.1 wt % Tween-20 and 0.1 U / μL ribonuclease inhibitor.

[0011] Furthermore, the in situ hybridization using RNAscope technology in step (3) is specifically as follows: S1, adding RNA probe to the tissue section of step (2) for incubation, and then washing; S2, adding a probe for subsequent signal amplification to the product obtained in step S1, incubating, and then washing; S3, adding a probe with an HRP label to the product obtained in step S2 for incubation, developing with a color developing solution, and then washing; S4, repeat steps S1-S3 according to the amount of target RNA; S5, sealing the product obtained in step S4 with a sealing agent and then imaging; S6, placing the tissue slice imaged in step S5 in a quenching reagent for fluorescence quenching; S7, placing the tissue slice quenched in step S6 in a probe elution reagent for elution.

[0012] Furthermore, the probes used for subsequent signal amplification in step S2 are AMP1, AMP2 and AMP3; the probes with HRP labels in step S3 are C1-HRP, C2-HRP and C3-HRP; the color development solution used in step S3 is C1-HRP developed with 488 color development solution; C2-HRP developed with cy3 color development solution; C3-HRP developed with cy5 color development solution.

[0013] Furthermore, the quenching reagent in step S5 is IRISKit HyperView Quench Buffer; and the probe elution reagent in step S6 includes the following components: 0.5 wt % ethylphenyl polyethylene glycol, 2×SSC and 30 v / v % formamide.

[0014] Furthermore, step (4) of protein immunohistochemical staining is specifically as follows: S1, adding a primary antibody and a secondary antibody to the product obtained in step (3) in sequence for incubation, then developing with a color developing solution, and finally eluting with an antibody eluting solution; wherein the primary antibody is Pancad, CD31 or NPHS2; the secondary antibody is HRP-coupled goat anti-rabbit IgG, HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG; the color developing solution is 488 color developing solution, cy3 color developing solution or cy5 color developing solution; S2, repeat step S1 according to the amount of target protein; S3. Seal the product obtained in step S2 with a sealing agent, and then image it.

[0015] The present invention has the following beneficial effects: (1) The multi-index RNA-protein co-staining method of the present invention combines the advantages of RNA in situ hybridization and protein immunohistochemical staining, not only providing RNA expression information, but also revealing the spatial localization and expression pattern of cellular proteins. Through the improvement of the co-staining method and the development of multiple reagents, a method for co-staining multiple target RNAs and target proteins on the same tissue section is realized, overcoming the protein antibody species restrictions and RNA non-specific staining problems encountered in the existing super-multi-index co-staining methods, and realizing the use of RNA to define cell subpopulations in spatial proteomics, and to visualize and locate all target cells of interest on the entire tissue section. It breaks through the limitations of traditional single detection methods and provides researchers with a comprehensive analysis of immune cells, tumor cells and other cell types in the tumor microenvironment. This provides a powerful tool for in-depth understanding of the complexity of the tumor immune microenvironment, evaluating treatment responses and exploring new therapeutic targets.

[0016] (2) In the in situ hybridization technique, protein digestion is required after antigen repair. The role of protease digestion is to remove proteins in cells, reduce possible background noise, and reduce non-specific binding of probes to non-target proteins in cells, thereby improving the binding efficiency of probes to target nucleic acids. However, after protease digestion, some proteins in the tissue sample will be partially or completely digested, which may cause subsequent protein staining to fail to proceed smoothly. The probe penetrating reagent of the present invention does not involve protein digestion, and can improve the permeability of the cell membrane, helping the probe to enter the cell smoothly. At the same time, it will not destroy the proteins inside and outside the cell, ensuring the integrity of the protein and ensuring that subsequent protein staining can proceed smoothly. Therefore, the probe penetrating reagent of the present invention can effectively improve the penetration efficiency of the probe, so that nucleic acid hybridization and protein staining can be carried out simultaneously, enhancing the sensitivity and specificity of the experiment.

[0017] (3) The present invention has developed a new type of highly efficient probe blocking reagent, which can effectively and completely block sites in tissue samples that may produce non-specific binding, thereby reducing background signals. Ethylene carbonate is added to the probe blocking reagent of the present invention, which can increase the affinity of the short DNA sequence used for blocking with all possible binding sites on the tissue section. Therefore, the probe blocking reagent of the present invention can prevent the non-specific binding of the probe to these sites by occupying non-target binding sites. When the probe binds to the target sequence with high specificity, the high concentration of the probe can displace the short fragments in the blocking reagent that bind to the non-target area, ensuring the clarity and accuracy of the signal.

[0018] (4) The spatial distribution of different RNA molecules in cells is usually at the micron level, and the specific spacing depends on the type of RNA, the cell type, and the dynamic process in the cell. If only spectral splitting is used to achieve multi-color labeling, cross-color may occur, resulting in the inability to accurately separate and identify different RNA signals. Therefore, the present invention selects three fluorescent channels with a long spectral distance (for example, 488, Cy3, and Cy5) for labeling. Through such a combination, three RNAs can be labeled at one time. After imaging, the three fluorescent labels are quenched using light quenching technology, thereby clearing the previous labels and preparing space for the next round of labeling, and performing the next round of three labels. Through this light cycle process, super-multiple staining of RNA can be achieved.

[0019] (5) RNAscope technology uses tyramide signal amplification technology to amplify signals, which makes the color reaction of each RNA signal more significant. In each cycle, three fluorescent channels correspond to three different HRP-labeled probes. However, since the same HRP-labeled probe is used in each round, if it is not adequately cleared, the signal labeled in the previous round may affect the color reaction in the next round. Therefore, in addition to light quenching, it is also necessary to use probe elution reagent to remove the previous labeled probes after each round of imaging, including target binding probes, AMP1, AMP2, AMP3, C1-HRP, C2-HRP, C3-HRP, etc. This step can effectively avoid signal interference and ensure that each round of labeling and color development can be carried out cleanly and accurately.

[0020] (6) The present invention combines light recycling technology with RNA elution reagents, which can not only avoid the interference of background signals during multiple staining, but also ensure efficient and clear resolution of the spatial distribution of multiple RNA molecules, greatly improving the sensitivity and accuracy of intracellular RNA expression research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the final effect of 9 labels and 10 colors achieved by co-staining 3 target RNAs and 6 target proteins in the multi-index RNA-protein co-staining method of the embodiment; Figure 2 The results of the effect of background quenching on signal detection before and after human placental tissue slices are shown in Figure A; Figure A is an image of a human placental tissue slice without background quenching treatment; Figure B is an image of a human placental tissue slice after background quenching treatment; Figure 3The final results of in situ hybridization of human ovarian cancer tissue sections without any permeabilization treatment, after treatment with probe penetrating reagent and after treatment with pepsin are shown in Figure A; Figure B is an image of human ovarian cancer tissue sections treated with pepsin digestion; Figure C is an image of human ovarian cancer tissue sections treated with probe penetrating reagent; Figure 4 The results of the effects of different blocking reagents on the target RNA signal of human placental tissue slices are shown in Figure 1; wherein Figure A is an image of a human placental tissue slice that has not been treated with a blocking reagent; Figure B is an image of a human placental tissue slice that has been treated with a blocking reagent that does not contain ethylene carbonate; and Figure C is an image of a human placental tissue slice that has been treated with the blocking reagent of the present invention; Figure 5 The results of the effect of probe elution on the in situ hybridization fluorescence signal are shown in Figure A. TIGB was labeled with a C2-HRP probe and developed with Cy3; Figure B was labeled with a C2-HRP probe and developed with Cy3; Figure C was an overlay of Figure A and Figure B after alignment with the DAPI signal using the IRRISS overlay software. DETAILED DESCRIPTION

[0022] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0023] Example:

[0024] A multi-index RNA-protein co-staining method comprises the following steps: (1) Sample preparation: Human kidney tissue sections were fixed with 4% paraformaldehyde (PFA) for 12 h and then embedded in paraffin; (2) Sample pretreatment (background quenching): paraffin-embedded human kidney tissue sections were dewaxed in 100wt% xylene for 3 times, 5 min each time; then dehydrated in anhydrous ethanol for 3 times, 3 min each time; then quenched with quenching reagent (IRISKit HyperView Quench Buffer) and placed in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 min; after quenching, washed with pure water 3 times, 1 min each time; then preheated the steamer to boiling, and heated the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appeared, then placed the human kidney tissue sections treated as above in the antigen repair solution and placed in a steamer for 15 min; then washed with 1× PBS buffer 3 times, 2 min each time, and finally circled the tissue with an oil pen; (3) Sample permeabilization treatment: add probe penetration reagent (1 wt% Tritonx-100 dissolved in 1×PBS) to the human kidney tissue slice treated in step (2) until it covers the entire human kidney tissue slice, and treat for 20 min; then observe the state of cells in the human kidney tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer for 3 times, each time for 1 min; (4) Tissue blocking treatment: add tissue blocking reagent to the human kidney tissue section of step (3) until the entire tissue section is covered, block at 40°C for 60 minutes, then wash twice with PBSTR solution, 2 minutes each time; and then wash with sodium citrate buffer (2×SSC) for 1 minute; wherein the tissue blocking reagent comprises the following components: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate and 1 mg / mL herring sperm DNA; the PBSTR solution comprises the following components: 1×PBS, 0.1 wt% Tween-20 and 0.1 U / µL RNase inhibitor; (5) RNA in situ hybridization: using RNAscope® MµLtiplex Fluorescent Reagent Kit / RNAscope ® Multiplex Fluorescence Assay Kit (Advanced Cell Diagnostics, Inc., ACD, USA). The experimental steps were carried out according to the instructions of the kit (see: https: / / acdbio.com / rnascope-mµLtiplex-fluorescent-v2-assay). The specific steps are as follows: 1. Add RNA probe (NOTUM) to the human kidney tissue slices in step (4) and incubate at 40°C for 2 h; then wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min; then wash with 2×SSC for 1 min; 2. Incubate the three probes AMP1, AMP2 and AMP3 for subsequent signal amplification at 40°C in sequence. The specific operation is as follows: ① After incubating with AMP1 for 30 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 minutes, and then wash with 2×SSC for 1 minute; ②After incubating with AMP2 for 30 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 minutes, and then wash with 2×SSC for 1 minute; ③After incubating with AMP3 for 15 minutes, wash twice with the washing solution provided in the kit (PBSTR can be replaced with the above-mentioned washing solution), each time for 2 minutes, and then wash with 2×SSC for 1 minute; 3. Incubate the three HRP-labeled probes C1-HRP, C2-HRP and C3-HRP in sequence at 40°C and develop the color. The specific operations are as follows: ① After incubating with C1-HRP for 15 minutes, wash twice with the washing solution in the kit (which can be replaced with the PBSTR used for washing) for 2 minutes each time; then wash with 2×SSC for 1 minute; then develop the color at room temperature with 488 color developing solution for 8 minutes; finally wash twice with 2×SSC for 2 minutes each time; ②After C2-HRP incubation for 15 minutes, wash twice with the washing solution in the kit (PBSTR can be replaced with the above-mentioned washing solution), 2 minutes each time; then wash once with 2×SSC, 1 minute; then use cy3 color developing solution to develop color at room temperature for 8 minutes, and finally wash twice with 2×SSC, 2 minutes each time; ③After incubating with C3-HRP for 15 minutes, wash twice with the washing solution in the kit (which can be replaced with the PBSTR used for washing) for 2 minutes each time; then wash once with 2×SSC for 1 minute; then develop with cy5 color developing solution at room temperature for 8 minutes, and finally wash twice with 2×SSC for 2 minutes each time; ④ Incubate with DAPI for 5 min, wash twice with 2×SSC, 2 min each time; 4. Seal the tissue sections obtained in step 3 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging (if there are other target RNAs that need to be labeled and stained, repeat steps 1-3); 5. Soak the tissue slices imaged in step 4 in 1×PBS buffer for 10 minutes, wait for the cover slip to slide off automatically, and then wash with 1×PBS buffer for 3 times; then place the tissue slices in the quenching reagent (IRISKit HyperView Quench Buffer) and put them in the quenching instrument for fluorescence quenching for 10 minutes; 6. Place the tissue slices quenched in step 5 in a preheated probe elution reagent and elute at 60°C for 3 min; then place the tissue slices in a preheated 2×SSC and wash at 37°C for 3 min; wherein the probe elution reagent comprises the following components: 0.5wt% ethylphenyl polyethylene glycol (NP-40), 2×SSC buffer and 30 v / v% formamide; (6) Protein immunohistochemical staining: 1. The human kidney tissue sections after quenching and elution in step (5) were incubated and stained with antibodies at 30°C in sequence. The specific operation is as follows: ① Add 200µL of primary antibody Pancad to the tissue section and incubate for 20 minutes, then rinse with 1×PBS, then add 200µL of HRP-conjugated goat anti-rabbit IgG (H+L) antibody and incubate for 20 minutes, then rinse with 1×PBS, use 488 colorimetric solution for 5 minutes, then use antibody elution solution to elute at 60°C for 20 minutes, and then rinse with 1×PBS; ② Add 200µL of primary antibody CD31 to the tissue section and incubate for 20 minutes, then rinse with 1×PBS, then add 200µL of HRP-conjugated goat anti-mouse IgG (H+L) antibody and incubate for 20 minutes, then rinse with 1×PBS, use cy3 colorimetric solution for 5 minutes, then use antibody elution solution (IRISKit HyperViewAdvanced Ab-Stripping Kit) to elute at 60℃ for 20 minutes, and rinse with 1×PBS; ③ Add 200µL of primary antibody NPHS2 to the tissue section and incubate for 20 minutes, then rinse with 1×PBS, then add 200µL of HRP-conjugated goat anti-rabbit IgG (H+L) antibody and incubate for 20 minutes, then rinse with 1×PBS, use cy5 colorimetric solution for 5 minutes, then use antibody elution solution to elute at 60℃ for 20 minutes, and rinse with 1×PBS; 2. Seal the tissue sections obtained in step 1 with sealing medium (Biyuntian P0126), and then use confocal (Olympus spin) 20× large-area stitching imaging (if there are other target proteins that need to be labeled and stained, repeat step 1).

[0025] In this example, three target RNA markers were first completed, and then six target protein markers were completed. Finally, the three rounds of images were superimposed using IRRISS overlay software (Chengdu Minghong Tiancheng Biotechnology Co., Ltd., http: / / 172.16.16.230 / ) to present a 9-marker 10-color multiple staining image of multi-index RNA (3 markers)-protein (6 markers) co-stained (see Figure 1, among which POLR2A (RNA Polymerase II Subunit A): is the catalytic subunit of RNA polymerase II, responsible for transcribing the DNA template into pre-mRNA; PPIB (Peptidyl-Prolyl cis-trans Isomerase B): cyclophilin B, a key protein folding auxiliary enzyme; UBC (Ubiquitin C): is one of the genes encoding ubiquitin; DAPI (4',6-diamidino-2-phenylindole): a fluorescent dye; Pan-Cadherin: belongs to type I classical cadherin, is a transmembrane glycoprotein that mediates calcium-dependent cell-cell adhesion; NPHS2 (Nephrosis 2): podocyte slit protein; AQP1 (Aquaporin 1): water channel protein 1; Calbindin: calcium binding protein; CD31 (Cluster of Differentiation 31): platelet-endothelial cell adhesion molecule 1; CD8 (Cluster of Differentiation 8): is a key glycoprotein on the surface of T lymphocytes). Figure 1 It can be seen that the staining method of the present invention can be used to stain and mark multiple RNAs and proteins at the same time (the color can be modified in image processing software such as Qupath, Olympus VIA, and colored according to one's own needs. The original image contains specific information of each channel, and the labeling status of each target RNA or protein can be understood by viewing a single channel).

[0026] Test example: (1) Effect of background quenching on signal detection before and after tissue slices: The fluorescence signals of human placental tissue slices with and without background quenching were detected respectively. The specific operations are as follows: A. No background quenching: 1. Sample preparation: Human placental tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 hours and then embedded in paraffin; 2. Sample permeabilization treatment: Add probe penetration reagent (1wt% Tritonx-100 dissolved in 1×PBS) to the tissue slice treated in step 1 until it covers the entire tissue sample and treat for 20 minutes; then observe the cell status of the tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer 3 times, 1 minute each time; 3. Tissue blocking treatment: add tissue blocking reagent to the tissue section of step 2 until the entire tissue sample is covered, block at 40°C for 60 minutes, then wash twice with PBSTR solution, 2 minutes each time; then wash with sodium citrate buffer (2×SSC) for 1 minute; wherein the tissue blocking reagent includes the following components: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate and 1 mg / mL herring sperm DNA; PBSTR solution includes the following components: 1×PBS, 0.1wt% Tween-20 and 0.1U / µL RNase inhibitor; 4. RNA in situ hybridization: RNAscope® MµLtiplex Fluorescent Reagent Kit / RNAscope® Multiple Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., ACD, USA) was used. The specific experimental steps were carried out according to the instructions of the kit (see: https: / / acdbio.com / rnascope-mµLtiplex-fluorescent-v2-assay). The specific steps are as follows: ① Add RNA probe (NOTUM) to the tissue sections in step 3 and incubate at 40°C for 2 hours; then wash twice with the washing solution in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 minutes; then wash with 2×SSC for 1 minute; ② Incubate the three probes AMP1, AMP2 and AMP3 for subsequent signal amplification at 40°C in sequence. The specific operation is as follows: i. After incubating with AMP1 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for 1 min; ii. After incubating with AMP2 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for min; iii. After incubating with AMP3 for 15 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the PBSTR used for washing above), each time for 2 minutes, and then wash with 2×SSC for 1 minute; 3. Incubate the HRP-labeled probe C1-HRP at 40°C and develop color. The specific steps are as follows: i. After incubating with C1-HRP for 15 minutes, wash twice with the washing solution provided in the kit (PBSTR can be used as a replacement for washing) for 2 minutes each time; then wash with 2×SSC for 1 minute; then develop the color at room temperature with 488 color developing solution for 8 minutes; finally wash twice with 2×SSC for 2 minutes each time; ii. Incubate with DAPI for 5 min, wash twice with 2×SSC, 2 min each time; 4. Seal the tissue sections obtained in step 3 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0027] B. After background quenching treatment: 1. Sample preparation: Human placental tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 hours and then embedded in paraffin; 2. Background quenching: Dewax the tissue sections in step 1 in 100wt% xylene for 3 times, 5 minutes each time; then dehydrate in anhydrous ethanol for 3 times, 3 minutes each time; then place in a quenching reagent (IRISKitHyperView Quench Buffer), and then place in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 minutes; after quenching, wash with pure water for 3 times, 1 minute each time; then preheat the steamer to boiling, and heat the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appear, then place the tissue sections treated above in the antigen repair solution and place in a steamer for 15 minutes; then wash with 1×PBS buffer for 3 times, 2 minutes each time, and finally circle the tissue with an oil pen; 3. Sample permeabilization treatment: add probe penetration reagent (1wt% Tritonx-100 dissolved in 1×PBS) to the tissue slice treated in step 2 until the entire tissue sample is covered, and treat for 20 minutes; then observe the cell state of the tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer 3 times, 1 minute each time; 4. Tissue blocking treatment: add tissue blocking reagent to the tissue section of step 3 until the entire tissue sample is covered, block at 40°C for 60 minutes, then wash twice with PBSTR solution, 2 minutes each time; then wash with sodium citrate buffer (2×SSC) for 1 minute; wherein the tissue blocking reagent includes the following components: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate and 1 mg / mL herring sperm DNA; PBSTR solution includes the following components: 1×PBS, 0.1wt% Tween-20 and 0.1U / µL RNase inhibitor; 5. RNA in situ hybridization: RNAscope® MµLtiplex Fluorescent Reagent Kit / RNAscope® Multiple Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., ACD, USA) was used. The specific experimental steps were carried out according to the instructions of the kit (see: https: / / acdbio.com / rnascope-mµLtiplex-fluorescent-v2-assay). The specific steps are as follows: ① Add RNA probe (NOTUM) to the tissue section in step (4) and incubate at 40°C for 2 h; then wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min; then wash with 2×SSC for 1 min; ② Incubate the three probes AMP1, AMP2 and AMP3 for subsequent signal amplification at 40°C in sequence. The specific operation is as follows: i. After incubating with AMP1 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for 1 min; ii. After incubating with AMP2 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for min; iii. After incubating with AMP3 for 15 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the PBSTR used for washing above), each time for 2 minutes, and then wash with 2×SSC for 1 minute; ③Then incubate the HRP-labeled probe C1-HRP at 40°C and develop the color. The specific steps are as follows: i. After incubating with C1-HRP for 15 minutes, wash twice with the washing solution provided in the kit (PBSTR can be used as a replacement for washing) for 2 minutes each time; then wash with 2×SSC for 1 minute; then develop the color at room temperature with 488 color developing solution for 8 minutes; finally wash twice with 2×SSC for 2 minutes each time; ii. Incubate with DAPI for 5 min, wash twice with 2×SSC, 2 min each time; ④ Seal the tissue sections obtained in step ③ with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0028] Depend on Figure 2 As shown in Figure A, human placental tissue sections without background quenching treatment showed strong background fluorescence in the interstitial region, resulting in a relative weakening of the target RNA signal and a reduction in imaging effect; Figure 2 (B), the background fluorescence in the interstitial region disappears significantly and no longer interferes with the detection of the target RNA signal, thereby significantly improving the imaging effect and the clarity of the experimental results.

[0029] (2) Penetration effect of probe penetrating reagent on tissue sections: In the prior art, pepsin is often used to digest samples to help the hybridization reaction system (such as probes and buffer) to penetrate cells smoothly and enter the location of the target nucleic acid. Therefore, the probe penetrating reagent and pepsin of the present invention are used to treat human ovarian cancer tissue sections, and the specific operations are as follows: A. Treated with pepsin: 1. Sample preparation: Human ovarian cancer tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 h and embedded in paraffin; 2. Background quenching: Dewax the tissue sections in step 1 in 100wt% xylene for 3 times, 5 minutes each time; then dehydrate in anhydrous ethanol for 3 times, 3 minutes each time; then place in a quenching reagent (IRISKitHyperView Quench Buffer), and then place in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 minutes; after quenching, wash with pure water for 3 times, 1 minute each time; then preheat the steamer to boiling, and heat the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appear, then place the tissue sections treated above in the antigen repair solution and place in a steamer for 15 minutes; then wash with 1×PBS buffer for 3 times, 2 minutes each time, and finally circle the tissue with an oil pen; 3. Sample digestion: Digest the tissue sections from step 2 with pepsin solution for 30 min; then rinse twice with 1× PBS buffer; 4. DNA in situ hybridization: Use the probe in the human HER2 gene amplification detection kit (fluorescence in situ hybridization method, Kanglu Bio) to drop 10μL on the tissue section, cover the slide with a cover glass, put it into the hybridizer, and hybridize at 40℃ for 2h. After removing the slide, wash it with the washing solution in the kit, then counterstain with DAPI for 5min, and then wash it twice with 2×SSC, 2min each time; 5. Seal the tissue sections obtained in step 4 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0030] B. Treated with probe penetrating reagent: 1. Sample preparation: Human ovarian cancer tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 h and embedded in paraffin; 2. Background quenching: Dewax the tissue sections in step 1 in 100wt% xylene for 3 times, 5 minutes each time; then dehydrate in anhydrous ethanol for 3 times, 3 minutes each time; then place in a quenching reagent (IRISKitHyperView Quench Buffer), and then place in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 minutes; after quenching, wash with pure water for 3 times, 1 minute each time; then preheat the steamer to boiling, and heat the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appear, then place the tissue sections treated above in the antigen repair solution and place in a steamer for 15 minutes; then wash with 1×PBS buffer for 3 times, 2 minutes each time, and finally circle the tissue with an oil pen; 3. Sample permeabilization treatment: add probe penetration reagent (1wt% Tritonx-100 dissolved in 1×PBS) to the tissue slice treated in step 2 until the entire tissue sample is covered, and treat for 20 minutes; then observe the cell state of the tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer 3 times, 1 minute each time; 4. DNA in situ hybridization: Use the probe in the human HER2 gene amplification detection kit (fluorescence in situ hybridization method, Kanglu Bio) to drop 10μL on the tissue section, cover the slide with a cover glass, put it into the hybridizer, and hybridize at 40℃ for 2h. After peeling off the slide, wash it with the washing solution in the kit, then counterstain with DAPI for 5min, and wash it twice with 2×SSC, 2min each time; 5. Seal the tissue sections obtained in step 4 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0031] Depend on Figure 3 As shown in Figure A, human ovarian cancer tissue sections without any permeabilization treatment did not show any signal after DNA in situ hybridization, indicating that the probe failed to effectively penetrate the cell nucleus; human ovarian cancer tissue sections treated with pepsin (see Figure 3 B) and human ovarian cancer tissue sections treated with probe penetrating reagents (see Figure 3 Figure C) shows obvious DNA signals, indicating that the probe can successfully enter the cell nucleus under both strategies. Figure 3 Compared with Figure B, Figure 3 The exposure time required for imaging in Figure C is shorter. It can be seen that the effect of the probe penetration reagent of the present invention is equivalent to the traditional pepsin digestion strategy and shortens the exposure time required for imaging.

[0032] (3) Effect of blocking reagent on target RNA signal: Human placental tissue sections were blocked using a blocking reagent that does not contain ethylene carbonate and a blocking reagent of the present invention, respectively. The specific operation is as follows: A. Treated with blocking reagents without ethylene carbonate: 1. Sample preparation: Human placental tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 hours and then embedded in paraffin; 2. Background quenching: Dewax the tissue sections in step 1 in 100wt% xylene for 3 times, 5 minutes each time; then dehydrate in anhydrous ethanol for 3 times, 3 minutes each time; then place in a quenching reagent (IRISKitHyperView Quench Buffer), and then place in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 minutes; after quenching, wash with pure water for 3 times, 1 minute each time; then preheat the steamer to boiling, and heat the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appear, then place the tissue sections treated above in the antigen repair solution and place in a steamer for 15 minutes; then wash with 1×PBS buffer for 3 times, 2 minutes each time, and finally circle the tissue with an oil pen; 3. Sample permeabilization treatment: add probe penetration reagent (1wt% Tritonx-100 dissolved in 1×PBS) to the tissue slice treated in step 2 until the entire tissue sample is covered, and treat for 20 minutes; then observe the cell state of the tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer 3 times, 1 minute each time; 4. Tissue blocking treatment: add a tissue blocking reagent that does not contain ethylene carbonate to the tissue section of step 3 until the entire tissue sample is covered, block at 40°C for 60 minutes, then wash twice with PBSTR solution, 2 minutes each time; then wash with sodium citrate buffer (2×SSC) for 1 minute; wherein, the tissue blocking reagent that does not contain ethylene carbonate includes the following components: 2×SSC and 1 mg / mL herring sperm DNA; the PBSTR solution includes the following components: 1×PBS, 0.1wt% Tween-20 and 0.1U / µL RNase inhibitor; 5. RNA in situ hybridization: RNAscope® MµLtiplex Fluorescent Reagent Kit / RNAscope® Multiple Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., ACD, USA) was used. The specific experimental steps were carried out according to the instructions of the kit (see: https: / / acdbio.com / rnascope-mµLtiplex-fluorescent-v2-assay). The specific steps are as follows: ① Add RNA probe (NOTUM) to the tissue sections in step 4 and incubate at 40°C for 2 hours; then wash twice with the washing solution in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 minutes; then wash with 2×SSC for 1 minute; ② Incubate the three probes AMP1, AMP2 and AMP3 for subsequent signal amplification at 40°C in sequence. The specific operation is as follows: i. After incubating with AMP1 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for 1 min; ii. After incubating with AMP2 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for min; iii. After incubating with AMP3 for 15 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the PBSTR used for washing above), each time for 2 minutes, and then wash with 2×SSC for 1 minute; ③ Incubate the HRP-labeled probe C1-HRP at 40°C and develop color. The specific steps are as follows: i. After incubating with C1-HRP for 15 minutes, wash twice with the washing solution provided in the kit (PBSTR can be used as a replacement for washing) for 2 minutes each time; then wash with 2×SSC for 1 minute; then develop the color with cy5 color developing solution at room temperature for 8 minutes; finally wash twice with 2×SSC for 2 minutes each time; ii. Incubate with DAPI for 5 min, wash twice with 2×SSC, 2 min each time; ④ Seal the tissue sections obtained in step 3 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0033] B. Treatment with blocking reagent containing ethylene carbonate: 1. Sample preparation: Human placental tissue sections were fixed with 4% paraformaldehyde (PFA) for 24 hours and then embedded in paraffin; 2. Background quenching: Dewax the tissue sections in step 1 in 100wt% xylene for 3 times, 5 minutes each time; then dehydrate in anhydrous ethanol for 3 times, 3 minutes each time; then place in a quenching reagent (IRISKitHyperView Quench Buffer), and then place in a quenching instrument (LUMINIRIS light cycle fluorescence quencher) for 8 minutes; after quenching, wash with pure water for 3 times, 1 minute each time; then preheat the steamer to boiling, and heat the antigen repair solution (2wt% EDTA antigen repair solution prepared with pure water) until small bubbles appear, then place the tissue sections treated above in the antigen repair solution and place in a steamer for 15 minutes; then wash with 1×PBS buffer for 3 times, 2 minutes each time, and finally circle the tissue with an oil pen; 3. Sample permeabilization treatment: add probe penetration reagent (1wt% Tritonx-100 dissolved in 1×PBS) to the tissue slice treated in step 2 until the entire tissue sample is covered, and treat for 20 minutes; then observe the cell state of the tissue slice under an inverted microscope. If many cells are highly transparent, the sample permeabilization treatment can be stopped; after stopping the treatment, wash with 1×PBS buffer 3 times, 1 minute each time; 4. Tissue blocking treatment: add tissue blocking reagent to the tissue section of step 3 until the entire tissue sample is covered, block at 40°C for 60 minutes, then wash twice with PBSTR solution, 2 minutes each time; then wash with sodium citrate buffer (2×SSC) for 1 minute; wherein the tissue blocking reagent includes the following components: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate and 1 mg / mL herring sperm DNA; PBSTR solution includes the following components: 1×PBS, 0.1wt% Tween-20 and 0.1U / µL RNase inhibitor; 5. RNA in situ hybridization: RNAscope® MµLtiplex Fluorescent Reagent Kit / RNAscope® Multiple Fluorescent Reagent Kit (Advanced Cell Diagnostics, Inc., ACD, USA) was used. The specific experimental steps were carried out according to the instructions of the kit (see: https: / / acdbio.com / rnascope-mµLtiplex-fluorescent-v2-assay). The specific steps are as follows: ① Add RNA probe (NOTUM) to the tissue sections in step 4 and incubate at 40°C for 2 hours; then wash twice with the washing solution in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 minutes; then wash with 2×SSC for 1 minute; ② Incubate the three probes AMP1, AMP2 and AMP3 for subsequent signal amplification at 40°C in sequence. The specific operation is as follows: i. After incubating with AMP1 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for 1 min; ii. After incubating with AMP2 for 30 min, wash twice with the washing solution provided in the kit (which can be replaced with the above-mentioned PBSTR), each time for 2 min, and then wash with 2×SSC for min; iii. After incubating with AMP3 for 15 minutes, wash twice with the washing solution provided in the kit (which can be replaced with the PBSTR used for washing above), each time for 2 minutes, and then wash with 2×SSC for 1 minute; ③Then incubate the HRP-labeled probe C1-HRP at 40°C and develop the color. The specific steps are as follows: i. After incubating with C1-HRP for 15 minutes, wash twice with the washing solution provided in the kit (PBSTR can be used as a replacement for washing) for 2 minutes each time; then wash with 2×SSC for 1 minute; then develop the color with cy5 color developing solution at room temperature for 8 minutes; finally wash twice with 2×SSC for 2 minutes each time; ii. Incubate with DAPI for 5 min, wash twice with 2×SSC, 2 min each time; ④ Seal the tissue sections obtained in step 3 with sealing medium (Biyuntian P0126); then use confocal (Olympus spin) 20× large-area stitching imaging.

[0034] The results are as follows Figure 4 As shown, the three groups of samples A, B, and C are the imaging results of the same area of ​​human placenta homologous continuous section tissue. The experimental settings are as follows: Group A was not blocked, Group B was treated with a blocking reagent without ethylene carbonate, and Group C was treated with a blocking reagent containing ethylene carbonate. After the RNA in situ hybridization experiment, each group of samples completed image acquisition under the same imaging parameters (CF405 channel: 500 ms, CF561 channel: 200 ms), and the image processing system was used to perform maximum background signal subtraction while retaining the RNA signal points. Figure 4 As shown in Figure A, human placental tissue sections that were not blocked showed significant nonspecific staining. Figure 4 As shown in Figures B and C, the human placental tissue sections treated with blocking treatment have effectively improved this phenomenon. It is worth noting that human placental tissue sections treated with blocking reagents containing ethylene carbonate show superior performance compared to human placental tissue sections treated with blocking reagents without ethylene carbonate: the background nonspecific signal is significantly reduced, and the target RNA signal presents a clearer point-like focusing feature, indicating that the homemade buffer can more effectively inhibit non-target binding through specific steric hindrance, which is more conducive to subsequent further analysis.

[0035] (4) Effect of probe elution on in situ hybridization fluorescence signal: Human kidney tissue sections were stained twice using the C2-HRP probe in the RNAscope® Multiplex Fluorescent Reagent Kit / RNAscope® Multiplex Fluorescent Kit (the treatment method is referred to step (5) of the example). The results are shown in Figure 2. Figure 5 As shown (among them, R1-cy3 is stained with 550 dye after the first round of staining; R2-cy3 is also stained with 550 dye after the second round of staining, and then processed by software to give it two different colors of red and green; ITGB (Integrin Beta): Integrin β; PPIB (PeptidylprolylIsomerase B): Peptidylprolyl cis-trans isomerase B). Figure 5 Middle panel A labeled TIGB using C2-HRP probe and developed with Cy3; Figure 5 In the middle panel B, PPIB was labeled using the same C2-HRP probe and Cy3 for color development; Figure 5 Figure C in the middle shows the results of two rounds of overlay after DAPI signal registration using IRRISS overlay software. Since the same C2-HRP probe is used in both the first round (R1) and the second round (R2), if the probe is not eluted, the R1 signal may appear during R2 color development, resulting in overlap of the two rounds of signals after overlay. However, Figure 5 There is no signal overlap in Figure C, indicating that probe elution is feasible and effective.

[0036] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A tissue sealing reagent, characterized in that: The following components were included at final concentrations: 100-200 mg / mL ethylene carbonate, 150-250 mg / mL dextran sulfate, 30-40 mg / mL sodium chloride, 2-5 mg / mL citric acid, 25-30 mg / mL trisodium citrate dihydrate, and 1-5 mg / mL herring sperm DNA.

2. The tissue sealing reagent according to claim 1, characterized in that: The following components were included at final concentrations: 150 mg / mL ethylene carbonate, 200 mg / mL dextran sulfate, 35.1 mg / mL sodium chloride, 2.1 mg / mL citric acid, 26.3 mg / mL trisodium citrate dihydrate, and 1 mg / mL herring sperm DNA.

3. Use of the tissue sealing reagent according to any one of claims 1 to 2 in a multi-index RNA-protein co-staining method.

4. A multi-index RNA-protein co-staining method, characterized in that: The following steps are involved: (1) Sample permeabilization treatment: add a probe penetrating reagent to the tissue section until the entire tissue sample is covered, and treat for 10-20 minutes; wherein the probe penetrating reagent is 1wt% Tritonx-100 dissolved in 1×PBS; (2) Tissue blocking treatment: drip the tissue blocking reagent described in any one of claims 1 to 2 onto the tissue section of step (1) until the entire tissue sample is covered, treat for 40 min to 60 min, and then wash with PBSTR solution and 2×SSC in sequence; (3) Perform RNA in situ hybridization: Use RNAscope technology to perform in situ hybridization; (4) Perform protein immunohistochemical staining.

5. The multi-index RNA-protein co-staining method according to claim 4, characterized in that: The tissue sample in step (1) is a human or animal tissue sample.

6. The multi-index RNA-protein co-staining method according to claim 4, characterized in that: The PBSTR solution in step (2) includes the following components: 1×PBS, 0.1 wt % Tween-20 and 0.1 U / μL ribonuclease inhibitor.

7. The multi-index RNA-protein co-staining method according to claim 4, characterized in that: The in situ hybridization using RNAscope technology described in step (3) is specifically as follows: S1, adding RNA probe to the tissue section of step (2) for incubation, and then washing; S2, adding a probe for subsequent signal amplification to the product obtained in step S1, incubating, and then washing; S3, adding a probe with an HRP label to the product obtained in step S2 for incubation, developing with a color developing solution, and then washing; S4, repeat steps S1-S3 according to the amount of target RNA; S5, sealing the product obtained in step S4 with a sealing agent and then imaging; S6, placing the tissue slice imaged in step S5 in a quenching reagent for fluorescence quenching; S7, placing the tissue slice quenched in step S6 in a probe elution reagent for elution.

8. The multi-index RNA-protein co-staining method according to claim 7, characterized in that: The probes used for subsequent signal amplification in step S2 are AMP1, AMP2 and AMP3; the probes with HRP labels in step S3 are C1-HRP, C2-HRP and C3-HRP; the color development with color developing solution in step S3 is that the C1-HRP is developed with 488 color developing solution; the C2-HRP is developed with cy3 color developing solution; and the C3-HRP is developed with cy5 color developing solution.

9. The multi-index RNA-protein co-staining method according to claim 7, characterized in that: The quenching reagent in step S5 is IRISKit HyperView Quench Buffer; the probe elution reagent in step S6 includes the following components: 0.5wt% ethylphenyl polyethylene glycol, 2×SSC and 30v / v% formamide.

10. The multi-index RNA-protein co-staining method according to claim 4, characterized in that: The protein immunohistochemical staining described in step (4) is specifically as follows: S1, adding a primary antibody and a secondary antibody to the product obtained in step (3) in sequence for incubation, then developing with a color developing solution, and finally eluting with an antibody eluting solution; wherein the primary antibody is Pancad, CD31 or NPHS2; the secondary antibody is HRP-coupled goat anti-rabbit IgG, HRP-coupled goat anti-mouse IgG or HRP-coupled goat anti-rabbit IgG; the color developing solution is 488 color developing solution, cy3 color developing solution or cy5 color developing solution; S2, repeat step S1 according to the amount of target protein; S3. Seal the product obtained in step S2 with a sealing agent, and then image it.

Images