Multiplex immunofluorescent staining method and kit for positioning and detecting synthesis level of newborn RNA (Ribonucleic Acid)

Abstract

The invention discloses a multiple immunofluorescence staining method and a kit for positioning and detecting the synthesis level of newborn RNA (Ribonucleic Acid), and relates to the technical field of multiple immunofluorescence detection. A brand-new multiple immunofluorescent staining method is determined by optimizing the slc12a3 antibody, the concentration of the LTL stock solution, the marker staining sequence, the marker-fluorescent dye pairing relation and the like. According to the method, dyeing and positioning detection of the synthesis level of the newborn RNA of the sample to be detected are realized, the blank of a technology for detecting the synthesis level of the newborn RNA through tissue positioning is filled, the synthesis level of the newborn RNA is detected by adopting multiple immunofluorescent dyeing, the expression conditions of the newborn RNA at different parts can be simultaneously displayed on one section, and the detection accuracy is high. The utilization value and the utilization rate of the sample slice are improved, and the tissue newborn RNA synthesis level can be more simply, conveniently, efficiently and accurately detected, so that the disease state is more accurately reflected.

Description

Technical Field

[0001] The present invention belongs to the technical field of multiple immunofluorescence detection, and in particular relates to a multiple immunofluorescence staining method and a kit for positioning and detecting the synthesis level of nascent RNA. Background Art

[0002] Click chemistry: Click chemistry is a synthetic concept first proposed by Sharpless in 1998 and subsequently refined. Its core concept is that synthetic chemistry should be guided by molecular function, enabling the rapid and reliable chemical synthesis of a wide variety of molecules through the facile assembly of small building blocks. The copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), discovered by his team in 2002, is one of the most iconic reactions and is considered the crown jewel of modern click chemistry.

[0003] EU staining: EU, the full name of which is 5'-ethynyluridine, is a special uridine nucleoside analog. During the RNA transcription process of biological molecules, it can replace the original uracil (U) and participate in the newly synthesized RNA molecules. This unique property makes EU an important research tool. The unique labeling property of EU is that it can react with azide biotin through a specific chemical reaction. As a result of this reaction, the newly synthesized RNA molecules are fluorescently stained, allowing scientists to intuitively observe and track the distribution of RNA. Through fluorescence detection, researchers can observe the dynamic changes of RNA in cells or tissues in real time. However, at present, EU staining to detect RNA synthesis is mainly used in the field of cell staining, and there is still a lack of convenient detection process steps for in vivo applications to detect RNA synthesis levels.

[0004] Compared to traditional fluorescence staining, flow cytometry, or immunohistochemistry, multiplex immunofluorescence staining can increase the rate of discovery of new biomarkers and therapeutic target pathways. This is because multiplex immunofluorescence staining can define and characterize the tissue microenvironment from three dimensions: qualitative, quantitative, and localized, providing richer data dimensions and deeper analytical capabilities. Although multiplex immunofluorescence staining has excellent performance in both qualitative and quantitative aspects of the tissue microenvironment, it has many limitations in actual operation.

[0005] Ribosome biogenesis (RiBi) refers to the process of ribosome formation. Inhibiting RiBi can significantly reduce nascent RNA synthesis. RiBi inhibition has achieved initial success in the study of various tumors, and RiBi reduction plays a key role in chronic aging-related diseases such as osteoporosis. Therefore, the development of a convenient multiplex immunofluorescence staining technique for localizing and detecting nascent RNA synthesis levels could more accurately characterize, quantify, and localize nascent RNA synthesis in tissues, potentially improving disease diagnosis and treatment. Summary of the Invention

[0006] To address the above-mentioned problems existing in the prior art, the present invention provides a multiple immunofluorescence staining method for locating and detecting the level of nascent RNA synthesis. This method can more simply, efficiently and accurately perform qualitative, quantitative and location detection of nascent RNA synthesis, thereby more accurately and effectively locating and observing the level of nascent RNA synthesis in tissues, significantly improving the efficiency of nascent RNA synthesis detection, and solving the problem that the prior art is difficult to accurately qualitate, quantify and locate the level of nascent RNA synthesis in organ tissues.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the objects of the present invention is to provide a multiple immunofluorescence staining method, which comprises the following steps:

[0009] S1, prepare the sample to be dyed;

[0010] S2. Prepare slc12a3 staining working solution, EU staining working solution and LTL staining working solution respectively, wherein the slc12a3 staining working solution includes slc12a3 primary antibody working solution and slc12a3 secondary antibody working solution;

[0011] S3, sequentially performing slc12a3 staining, EU staining, and LTL staining on the sample to be stained to obtain a stained sample;

[0012] S4. Counterstain and seal the slides with a fluorescence quenching mounting medium to obtain the sample to be tested.

[0013] Furthermore, the method for preparing the sample to be stained in step S1 includes: fixing and embedding the tissue treated with EU solution, then slicing it into sections with a thickness of 3-5 μm, and then baking it at 65-75° C. for 1.5-2.5 hours.

[0014] Furthermore, in step S2: the slc12a3 primary antibody working solution and the slc12a3 secondary antibody working solution are respectively obtained by diluting the slc12a3 primary antibody and the fluorescent secondary antibody at a ratio of 1:280-320; each milliliter of the EU staining working solution includes 930-940ul enzyme-free water, 45-55ul 10mM BTTAA, 8-12ul 2mM CuSO4, 2.5-3.5ul 0.1% azide Cy3 fluorescent dye and 8-10mg sodium ascorbate; the LTL staining working solution is obtained by diluting the LTL stock solution at a ratio of 1:80-120.

[0015] Furthermore, the primary antibody of slc12a3 includes a rabbit-derived polyclonal antibody; the fluorescent secondary antibody of slc12a3 includes a donkey anti-rabbit 647 fluorescent secondary antibody; the excitation wavelength of the fluorescent dye corresponding to the EU staining working solution is 550 nm; and the excitation wavelength of the fluorescent dye corresponding to the LTL staining working solution is 488 nm.

[0016] Furthermore, in step S3: the staining procedure of the slc12a3 staining includes, in sequence: dewaxing, antigen retrieval, neutralization, tissue permeabilization, blocking, primary antibody incubation, secondary antibody incubation, and PBS elution; the staining procedure of the EU staining includes, in sequence: incubation in EU staining working solution and permeabilization agent elution; the staining procedure of the LTL staining includes, in sequence: incubation in LTL staining working solution and PBS elution.

[0017] Furthermore, the dewaxing comprises: sequentially placing in xylene I for 28-32 minutes, xylene II for 28-32 minutes, anhydrous ethanol for 8-12 minutes, 95% ethanol for 4-6 minutes, 85% ethanol for 4-6 minutes, 75% ethanol for 4-6 minutes, 50% ethanol for 4-6 minutes, rinsing with tap water for 1-3 minutes, and rinsing with PBS 1-3 times;

[0018] The antigen repair comprises: boiling the antigen repair solution in a microwave for 14-16 minutes, and washing with PBS for 8-12 minutes;

[0019] The neutralization comprises: incubating with neutralization solution for 8-12 minutes, washing with PBS 1-3 times, each time for 4-6 minutes;

[0020] The permeabilization of the tissue comprises: incubating the permeabilized tissue with a permeabilizing agent for 18-22 minutes, and rinsing with PBS for 8-12 minutes;

[0021] The blocking step includes: incubating in a blocking solution for 0.8-1.2 hours, washing with PBS 4-6 times, each time for 4-6 minutes;

[0022] The primary antibody incubation includes: incubating in the primary antibody working solution at 3-5° C. for 15.5-16.5 hours, washing with PBS 2-4 times, each time for 2-4 minutes;

[0023] The secondary antibody incubation includes: incubating the secondary antibody working solution at room temperature for 0.8-1.2 hours, washing with PBS 2-4 times, each time for 2-4 minutes;

[0024] The EU staining comprises: incubating the EU staining working solution in the dark at room temperature for 28-32 minutes, and washing with the penetrant 2-4 times, each time for 8-12 minutes;

[0025] The LTL staining comprises: incubating the LTL staining working solution in the dark at room temperature for 2.5-3.5 hours, washing with PBS 4-6 times, each time for 2-4 minutes;

[0026] The antigen repair solution includes an improved sodium citrate antigen repair solution;

[0027] The neutralization solution includes 1.8-2.2 mg / ml glycine solution;

[0028] The penetrant includes 0.4-0.6% TritonX-100 solution;

[0029] The blocking solution includes 4-6% BSA solution.

[0030] Furthermore, the fluorescence quenching mounting medium in step S4 includes an anti-fluorescence quenching mounting medium containing Dapi.

[0031] A second object of the present invention is to provide a multiplex immunofluorescence detection method, which comprises using any of the multiplex immunofluorescence staining methods described above and the reagents used therein, and also comprises the steps of microscopic imaging, image analysis, and obtaining nascent RNA synthesis level analysis data.

[0032] A third object of the present invention is to provide an application of any of the above-mentioned multiple immunofluorescence staining methods in the preparation of a product for detecting the level of nascent RNA synthesis.

[0033] A fourth object of the present invention is to provide a product for detecting the level of nascent RNA synthesis, wherein the product comprises a kit containing the reagents used in any of the above-mentioned multiple immunofluorescence staining methods.

[0034] Furthermore, the kit includes the following reagents: EU powder, antigen retrieval solution, neutralizing solution, tissue permeabilization agent, blocking solution, antibody diluent, anti-slc12a3 primary antibody, fluorescent secondary antibody, enzyme-free water, BTTAA, CuSO4, 0.1% azide Cy3 fluorescent dye, sodium ascorbate, LTL stock solution, PBS, and Dapi-containing anti-fluorescence quenching mounting medium.

[0035] Furthermore, the antigen retrieval solution includes an improved sodium citrate antigen retrieval solution; the anti-slc12a3 primary antibody includes a rabbit-derived polyclonal antibody; and the fluorescent secondary antibody includes a donkey anti-rabbit 647 fluorescent secondary antibody.

[0036] A fifth object of the present invention is to provide the use of any of the above-mentioned multiple immunofluorescence staining methods and / or the products (kits) in the preparation of renal fibrosis detection products.

[0037] Compared with the prior art, the present invention has the following technical effects or advantages:

[0038] 1. The present invention proposes a multiplex immunofluorescence staining method for localizing and detecting the level of nascent RNA synthesis. This method achieves staining and localization detection of nascent RNA synthesis levels in the sample to be tested by selecting appropriate reagents and a reasonable staining procedure. The use of multiplex immunofluorescence staining to detect nascent RNA synthesis levels can simultaneously display the expression of nascent RNA in different parts of a single slice. Compared with the existing EU staining method for detecting nascent RNA synthesis in cells, the present invention not only achieves the detection of nascent RNA synthesis levels in tissues, but also provides a method for detecting its expression localization, thereby increasing the utilization value of sample slices, especially rare samples.

[0039] 2. Existing EU staining is limited to the cellular level, suffers from poor tissue penetration (EU requires a permeabilizing agent for tissue localization), lacks anatomical reference markers, and sequential staining easily leads to fluorescence interference. This present invention overcomes these obstacles by proposing a slc12a3 / EU / LTL triple marker sequential staining method, filling the gap in tissue localization detection of nascent RNA synthesis levels.

[0040] 3. The multiple immunofluorescence staining method proposed in the present invention is used to locate and detect the level of nascent RNA synthesis. The stained samples can be imaged using an ultra-high-resolution laser confocal microscope, and the images obtained can be analyzed using ImageJ image analysis software. The detection and analysis process is simple, reliable, and easy to operate, and has broad application prospects.

[0041] 4. The present invention proposes a multiplex immunofluorescence staining kit for localizing and detecting the synthesis level of nascent RNA. The reagents in the kit can be used for staining and localizing the synthesis level of nascent RNA in samples. In particular, LTL and anti-slc12a3 polyclonal antibodies are used for the first time in nascent RNA detection, greatly improving the accuracy and efficiency of detection, thereby more accurately reflecting the disease status.

[0042] 5. The multiplex immunofluorescence detection method proposed in the present invention for localizing and detecting the level of nascent RNA synthesis combines the azide reaction with a localization marker to explore the optimal detection method, clearly provides the experimental steps for how to detect nascent RNA in animal tissues, and realizes how to achieve in situ, multiplex, and quantitative localization analysis of nascent RNA synthesis in organ tissues. It breaks through the limitation of existing technologies that are limited to cellular level detection, solves the problem of analyzing spatial heterogeneity in tissue microenvironments, and provides a new approach for efficient and rapid detection of nascent RNA at the tissue level. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the process of positioning and detecting the synthesis level of nascent RNA in the present invention.

[0044] Figure 2 Schematic diagram of the multiple immunofluorescence staining process in the present invention.

[0045] Figure 3 Comparison of simple EU staining and multiplex immunofluorescence staining of samples 1-3.

[0046] Figure 4 Comparison of simple EU staining and multiplex immunofluorescence staining of samples 4-6.

[0047] Figure 5 Comparison of simple EU staining and multiplex immunofluorescence staining of samples 7-10.

[0048] Figure 6 Statistical analysis of simple EU staining and multiple immunofluorescence staining of samples 1-10.

[0049] Figure 7 Comparison of multiple immunofluorescence staining and double immunofluorescence staining of samples 1-3.

[0050] Figure 8 Comparison chart of multiple immunofluorescence staining and double immunofluorescence staining of samples 4-6.

[0051] Figure 9 The staining comparison images of normal mouse kidney tissue and renal fibrosis mouse kidney tissue samples 1-3.

[0052] Figure 10 This is a staining comparison of samples 4-6 of normal mouse kidney tissue and renal fibrosis mouse kidney tissue.

[0053] Figure 11 This is a statistical analysis chart showing the staining comparison of kidney tissue samples from normal mice and mice with renal fibrosis. DETAILED DESCRIPTION

[0054] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the inventive method, steps or conditions are intended to fall within the scope of the present invention. The reagents, products and instruments used in the following examples are all commercially available, and the methods used in the examples are consistent with conventional methods unless otherwise specified.

[0055] The overall technical idea of ​​the present invention is: Fluorescence resonance energy transfer refers to the physical phenomenon of energy transfer from an excited fluorescent group to another fluorescent group. The former is called a donor and the latter is called an acceptor. When the two fluorescent dyes are close enough, fluorescence resonance energy transfer will occur. The excited donor transfers part of the energy to the acceptor in a non-radiative manner, causing the acceptor to be excited to emit fluorescence, while its own fluorescence is quenched. In order to avoid the occurrence of fluorescence resonance energy transfer, which leads to a poor final staining effect, the present invention optimizes the concentration of slc12a3 antibody and LTL stock solution (i.e., dilution multiple, see Table 1 for details), the marker staining sequence and the marker-fluorescent dye pairing relationship, and determines the optimal antibody concentration, marker staining sequence and marker-fluorescent dye pairing, which can effectively eliminate the staining problem caused by the fluorescence resonance energy transfer phenomenon.

[0056] The technical solution of the present invention is further elaborated in detail below in conjunction with embodiments.

[0057] Example 1

[0058] This embodiment provides a multiplex immunofluorescence detection method for detecting the level of nascent RNA synthesis in the kidney, comprising the following steps:

[0059] (1) Prepare the sample to be dyed: Figure 1 Schematic diagram of the process of positioning and detecting the synthesis level of nascent RNA in the present invention, as shown in FIG. Figure 1 The samples to be tested were processed as shown. All samples were injected intraperitoneally with EU solution 6 hours before sampling, and all tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin tissues were sliced ​​at a thickness of 3-5 μm and mounted on positively charged glass slides. All samples were stained with H&E to assess whether there was tissue damage. The prepared slices were placed in a 70°C oven for 2 hours to obtain the samples to be stained.

[0060] (2) Reagent preparation: dilute the primary antibody against slc12a3, a marker of the distal tubules of the kidney, with antibody diluent to obtain a primary antibody working solution; dilute the fluorescent secondary antibody against slc12a3, a marker of the distal tubules of the kidney, with antibody diluent to obtain a secondary antibody working solution; prepare an EU staining working solution using enzyme-free water, BTTAA, CuSO4, azide Cy3 fluorescent dye, and sodium ascorbate; dilute the stock solution of LTL, a marker of the proximal tubules of the kidney, with antibody diluent to obtain an LTL staining working solution; the main reagent information of this example is shown in Table 1.

[0061] (3) The sample to be stained is first stained with the distal tubule marker slc12a3, and the staining procedure includes: dewaxing, antigen retrieval, neutralization, tissue permeabilization, blocking, primary antibody incubation, secondary antibody incubation, and PBS elution; then EU staining is performed, and the staining procedure includes: EU staining working solution incubation, permeabilization agent elution; finally, the proximal tubule marker LTL is stained, and the staining procedure includes: LTL staining working solution incubation, PBS elution; the staining process is as follows Figure 2 As shown, the pairing relationship between markers and fluorescent dyes and the staining sequence are shown in Table 2, and the specific staining procedure is shown in Table 3 to obtain the stained samples.

[0062] (4) After the staining process is completed, the stained sample is taken out, and a Dapi anti-fluorescence quenching sealing agent is added dropwise for staining and the sample is sealed with a cover glass to obtain a sample to be tested.

[0063] (5) The sample to be tested can be imaged using an ultra-high-resolution laser confocal microscope, and the image obtained is analyzed using ImageJ image analysis software. Based on the co-localization of the EU staining results with the proximal tubule marker or the distal tubule marker, analytical data on the level of nascent RNA synthesis located in the proximal tubule or the distal tubule is obtained.

[0064] Table 1 Main reagent information

[0065]

[0066] Table 2 Relationship between markers and fluorescent dye excitation wavelength pairing and staining sequence

[0067]

[0068] Table 3 Staining procedure

[0069]

[0070] Note: Antigen retrieval solution is improved sodium citrate antigen retrieval solution; neutralization solution is 2 mg / ml glycine solution; tissue penetrant is 0.5% TritonX-100 solution; blocking solution is 5% BSA solution; washing solution is PBS solution.

[0071] Example 2

[0072] The purpose of this example is to compare and verify the effectiveness of the multiple immunofluorescence detection method of the present invention and the simple EU staining method.

[0073] Ten mouse kidney tissue samples were selected (mice were purchased from Alvin, C57BL / 6JNifdc male mice). Tissue sections were prepared for each sample according to step (1) of Example 1. One section was used for confocal microscopy imaging of multiple immunofluorescence staining of the present invention, and the other section was used for conventional fluorescence microscopy imaging of simple EU staining. The multiple immunofluorescence staining and analysis methods were referred to Example 1. The specific steps of the simple EU staining method are shown in Table 4. The images of multiple immunofluorescence staining and simple EU staining are shown in Table 4. Figures 3 to 5 , the staining statistical results are shown in Table 5. As shown in Table 5 and Figure 6 As shown in the figure, the effectiveness of multiple immunofluorescence staining is significantly higher than that of simple EU staining, and multiple immunofluorescence staining can also more intuitively locate and observe the level of nascent RNA synthesis based on the image.

[0074] Table 4 Simple EU staining procedure

[0075]

[0076] Table 5 Statistics of multiple immunofluorescence staining and simple EU staining results

[0077]

[0078] Example 3

[0079] The purpose of this example is to compare the staining effects between the multiple immunofluorescence detection method of the present invention and the double fluorescence staining method.

[0080] Six mouse kidney tissue samples were selected (mice were purchased from Alvatorevo, C57BL / 6JNifdc male mice). Three consecutive tissue sections were prepared for each sample according to step (1) of Example 1. One section was used for the confocal microscopy imaging of the multiple immunofluorescence staining of the present invention, and two sections were used for confocal microscopy imaging of the double immunofluorescence staining. One of the double immunofluorescence staining sections was used for staining the proximal tubule markers LTL and EU, and the other was used for staining the distal tubule markers slc12a3 and EU. The multiple immunofluorescence staining and analysis method was referred to Example 1. The specific steps of the double immunofluorescence staining method are shown in Tables 6 and 7. The pictures of the multiple immunofluorescence staining and double immunofluorescence staining are shown in Figure 7 and Figure 8 .like Figure 7 and Figure 8As shown in the results, multiple immunofluorescence staining can simultaneously display the synthesis levels of nascent RNA in the proximal tubules and distal tubules compared with double immunofluorescence staining, which significantly improves the sample utilization value of double immunofluorescence staining, saves the use of staining reagents, and saves staining costs.

[0081] Table 6 Double immunofluorescence staining procedure (LTL and EU staining)

[0082]

[0083] Table 7 Double immunofluorescence staining procedure (slc12a3 and EU staining)

[0084]

[0085] Example 4

[0086] The purpose of this example is to apply the multiple immunofluorescence detection method of the present invention to a renal fibrosis disease model, namely, a unilateral ureteral ligation model.

[0087] Six normal control mouse kidney tissue samples and six renal fibrosis mouse kidney tissue samples were selected (mice were purchased from Alvin, C57BL / 6JNifdc male mice; a unilateral ureteral ligation mouse model was constructed by ourselves: after the mice were anesthetized, the abdominal skin and muscle layer were cut open layer by layer along the midline of the abdomen. The left kidney of the mouse was exposed, the left ureter was found, and the ureter was ligated at two points with 5-0 thread. The abdominal organs were returned, and the abdominal muscle layer and skin tissue were sutured layer by layer with 5-0 thread. The abdominal skin wound was disinfected with iodine). For each sample, tissue sections were prepared for multiple immunofluorescence staining according to step (1) of Example 1. The staining method was referred to Example 1. The staining results are shown in FIG. Figure 9-10 Statistical analysis results are shown in Figure 11 The experimental results show that the detection kit provided by the present invention, when used in the detection of renal fibrosis, can not only visually locate and observe that the level of nascent RNA synthesis in the proximal tubules is significantly reduced, but statistical analysis also shows the same significant results, suggesting that the multiple immunofluorescence staining kit provided by the present invention for locating and detecting the level of nascent RNA synthesis has application prospects in the preparation of products for detecting renal fibrosis.

[0088] In summary, the multiple immunofluorescence staining method for localizing and detecting nascent RNA synthesis levels of the present invention realizes the staining and localization detection of nascent RNA synthesis levels in the sample to be tested, filling the gap in the technology of tissue localization detection of nascent RNA synthesis levels. By using multiple immunofluorescence staining to detect nascent RNA synthesis levels, the expression of nascent RNA in different parts of a slice can be simultaneously displayed, thereby improving the utilization value and utilization rate of sample slices, and can more simply, efficiently and accurately detect the nascent RNA synthesis level of tissues, thereby more accurately reflecting the disease status.

[0089] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A multiple immunofluorescence staining method, characterized in that: The following steps are involved: S1, prepare the sample to be dyed; S2. Prepare slc12a3 staining working solution, EU staining working solution and LTL staining working solution respectively, wherein the slc12a3 staining working solution includes slc12a3 primary antibody working solution and slc12a3 secondary antibody working solution; S3, sequentially performing slc12a3 staining, EU staining, and LTL staining on the sample to be stained to obtain a stained sample; S4. Counterstain and seal the slides with a fluorescence quenching mounting medium to obtain the sample to be tested.

2. The multiple immunofluorescence staining method according to claim 1, characterized in that The method for preparing the sample to be stained in step S1 includes: fixing and embedding the tissue treated with EU solution, then slicing it into sections with a thickness of 3-5 μm, and then baking it at 65-75° C. for 1.5-2.5 hours.

3. The multiple immunofluorescence staining method according to claim 1, wherein In step S2: The slc12a3 primary antibody working solution and the slc12a3 secondary antibody working solution are respectively obtained by diluting the slc12a3 primary antibody and the fluorescent secondary antibody at a ratio of 1:280-320; Each ml of the EU staining working solution includes 930-940 μl of enzyme-free water, 45-55 μl of 10 mM BTTAA, 8-12 μl of 2 mM CuSO 4 , 2.5-3.5 μl of 0.1% azide Cy3 fluorescent dye, and 8-10 mg of sodium ascorbate; The LTL staining working solution is obtained by diluting the LTL stock solution at a ratio of 1:80-120.

4. The multiple immunofluorescence staining method according to claim 3, characterized in that The primary antibody against slc12a3 includes a rabbit-derived polyclonal antibody; The fluorescent secondary antibody against slc12a3 includes donkey anti-rabbit 647 fluorescent secondary antibody; The excitation wavelength of the fluorescent dye corresponding to the EU staining working solution is 550nm; The excitation wavelength of the fluorescent dye corresponding to the LTL staining working solution is 488 nm.

5. The multiple immunofluorescence staining method according to claim 1, characterized in that: In step S3: The staining procedure of slc12a3 staining includes: dewaxing, antigen retrieval, neutralization, tissue permeabilization, blocking, primary antibody incubation, secondary antibody incubation, and PBS elution; The EU staining procedure includes: incubation with EU staining working solution, elution with penetrant; The staining procedure of the LTL staining includes: incubation with LTL staining working solution and elution with PBS.

6. The multiple immunofluorescence staining method according to claim 1, characterized in that: The fluorescence quenching mounting medium in step S4 includes a Dapi-containing anti-fluorescence quenching mounting medium.

7. A multiplex immunofluorescence detection method, characterized in that: The method comprises the steps of using the multiple immunofluorescence staining method according to any one of claims 1 to 6, and further comprises the steps of microscopic imaging, image analysis, and obtaining analytical data on the level of nascent RNA synthesis.

8. Use of the multiple immunofluorescence staining method according to any one of claims 1 to 6 in the preparation of a product for detecting nascent RNA synthesis levels.

9. A product for detecting the level of nascent RNA synthesis, characterized in that The product includes a kit comprising the reagents used in the multiple immunofluorescence staining method according to any one of claims 1 to 6.

10. Use of the multiple immunofluorescence staining method according to any one of claims 1 to 6 and / or the product according to claim 9 in the preparation of a product for detecting renal fibrosis.

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