A multiplex immunofluorescence staining method and kit for localizing and detecting the level of nascent RNA synthesis.

Abstract

This invention discloses a multiplex immunofluorescence staining method and kit for the localized detection of nascent RNA synthesis levels, belonging to the field of multiplex immunofluorescence detection technology. This invention optimizes the concentrations of slc12a3 antibody and LTL stock solution, the staining order of markers, and the marker-fluorescent dye pairing relationship to establish a novel multiplex immunofluorescence staining method. This method achieves staining and localized detection of nascent RNA synthesis levels in test samples, filling a gap in tissue-localized detection of nascent RNA synthesis levels. Using multiplex immunofluorescence staining to detect nascent RNA synthesis levels allows for simultaneous display of nascent RNA expression at different sites on a single tissue section, improving the utilization value and efficiency of sample sections. It enables simpler, more efficient, and accurate detection of tissue nascent RNA synthesis levels, thus more accurately reflecting disease states.

Description

Technical Field

[0001] This invention belongs to the field of multiplex immunofluorescence detection technology, and particularly relates to a multiplex immunofluorescence staining method and kit for localizing and detecting the level of nascent RNA synthesis. Background Technology

[0002] Click chemistry: Click chemistry is a synthetic concept initially proposed by Sharpless in 1998 and subsequently refined. Its core idea is that synthetic chemistry should be function-oriented, using the simple assembly of small units to rapidly and reliably synthesize a wide variety of molecules. The monovalent copper ion-catalyzed azide-alkyne cycloaddition reaction (CuAAC), discovered by his team in 2002, is one of the most representative reactions and is considered the crown jewel of modern click chemistry.

[0003] EU staining: EU, short for 5'-ethynyluridine, is a special uracil nucleoside analog. During RNA transcription in biomolecules, it can replace the original uracil (U) and participate in the newly synthesized RNA molecule. This unique property makes EU an important research tool. EU's unique labeling characteristic lies in its ability to react with azide-biotin through a specific chemical reaction. The result of this reaction is that the newly synthesized RNA molecule is fluorescently stained, allowing scientists to visually 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, currently, EU staining for RNA synthesis detection is mainly used in the field of cell staining; convenient detection procedures are still lacking for in vivo applications to detect RNA synthesis levels.

[0004] Compared to traditional fluorescence staining, flow cytometry, or immunohistochemistry, multiplex immunofluorescence staining can improve the speed of discovering new biomarkers and therapeutic targeting pathways. This is because multiplex immunofluorescence staining can define and characterize the tissue microenvironment from three dimensions: qualitative, quantitative, and localization, providing richer data dimensions and more in-depth analytical capabilities. Although multiplex immunofluorescence staining performs exceptionally well in the qualitative and quantitative analysis of the tissue microenvironment, it has many limitations in practical application.

[0005] Ribosome biogenesis (RiBi) refers to the process of ribosome formation. Inhibiting RiBi can significantly reduce the synthesis of newly synthesized RNA. Current research on RiBi inhibition has shown preliminary success in various neoplastic diseases, and the reduction of RiBi plays a crucial role in chronic age-related diseases such as osteoporosis. Therefore, developing a convenient multiplex immunofluorescence staining technique for the localization and detection of newly synthesized RNA synthesis levels is crucial. This technique could more accurately qualitatively, quantitatively, and locally locate the synthesis of newly synthesized RNA in tissues, potentially improving the diagnosis and treatment of diseases. Summary of the Invention

[0006] To address the aforementioned problems in the existing technology, this invention provides a multiplex immunofluorescence staining method for the localization and detection of nascent RNA synthesis levels. This method enables simpler, more efficient, and more accurate qualitative, quantitative, and localization detection of nascent RNA synthesis, thereby more accurately and effectively locating and observing the nascent RNA synthesis levels in tissues, significantly improving the efficiency of nascent RNA synthesis detection, and solving the problem that existing technologies are unable to accurately qualitatively, quantitatively, and locally locate the nascent RNA synthesis levels in organs and tissues.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] One objective of this invention is to provide a multiplex immunofluorescence staining method, the method comprising the following steps:

[0009] S1. Prepare the sample slides to be stained;

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

[0011] S3. The stained specimens are stained sequentially with slc12a3 staining, EU staining and LTL staining to obtain stained specimens.

[0012] S4. Restain with fluorescent quenching mounting medium and mount to obtain the sample to be tested.

[0013] Furthermore, the preparation method of the stained sample in step S1 includes: fixing and embedding the tissue treated with EU solution, then slicing it with a thickness of 3-5 μm, and then baking it at 65-75℃ for 1.5-2.5 h.

[0014] Further, in step S2: the slc12a3 primary antibody working solution and the slc12a3 secondary antibody working solution are obtained by diluting the slc12a3 primary antibody and the fluorescent secondary antibody at a ratio of 1:280-320, respectively; each milliliter of the EU staining working solution includes 930-940ul of enzyme-free water, 45-55ul of 10mM BTTAA, 8-12ul of 2mM CuSO4, 2.5-3.5ul of 0.1% azide Cy3 fluorescent dye, and 8-10mg of 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 for slc12a3 includes a rabbit-derived polyclonal antibody; the fluorescent secondary antibody for 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 for slc12a3 staining includes, in sequence: dewaxing, antigen retrieval, neutralization, tissue permeation, blocking, primary antibody incubation, secondary antibody incubation, and PBS elution; the staining procedure for EU staining includes, in sequence: EU staining working solution incubation and permeabilizer elution; the staining procedure for LTL staining includes, in sequence: LTL staining working solution incubation and PBS elution.

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

[0018] The antigen retrieval process includes: microwaving the antigen retrieval solution to boiling for 14-16 minutes, followed by rinsing with PBS for 8-12 minutes.

[0019] The neutralization process includes: incubation with neutralizing solution for 8-12 min, followed by washing with PBS 1-3 times, each time for 4-6 min.

[0020] The tissue permeation process includes: incubating the tissue permeation agent for 18-22 min, followed by rinsing with PBS for 8-12 min.

[0021] The blocking process includes: incubation with blocking solution for 0.8-1.2 h, followed by washing with PBS 4-6 times, each time for 4-6 min;

[0022] The primary antibody incubation includes: incubation in primary antibody working solution at 3-5℃ for 15.5-16.5h, followed by washing with PBS 2-4 times, each time for 2-4min;

[0023] The secondary antibody incubation includes: incubation in the secondary antibody working solution at room temperature for 0.8-1.2 h, followed by washing with PBS 2-4 times, each time for 2-4 min;

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

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

[0026] The antigen retrieval solution includes a modified sodium citrate antigen retrieval solution;

[0027] The neutralizing solution comprises a glycine solution of 1.8-2.2 mg / ml;

[0028] The penetrant comprises a 0.4-0.6% Triton X-100 solution;

[0029] The sealing solution comprises a 4-6% BSA solution.

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

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

[0032] The third objective of this invention is to provide the application of any of the above-described multiplex immunofluorescence staining methods in the preparation of products for detecting the level of nascent RNA synthesis.

[0033] The fourth objective of this invention is to provide a product for detecting the level of nascent RNA synthesis, the product comprising a kit containing reagents used in any of the above-described multiplex immunofluorescence staining methods.

[0034] Furthermore, the kit includes the following reagents: EU powder, antigen retrieval solution, neutralizing solution, tissue permeabilizer, 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 mounting medium containing Dapi anti-fluorescence quenching agent.

[0035] Furthermore, the antigen retrieval solution includes a modified 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] The fifth objective of this invention is to provide the application of any of the above-described multiplex 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 the localization detection of nascent RNA synthesis levels. This method, by selecting appropriate reagents and a reasonable staining procedure, achieves the staining and localization detection of nascent RNA synthesis levels in the sample to be tested. By using multiplex immunofluorescence staining to detect nascent RNA synthesis levels, the expression of nascent RNA in different sites can be simultaneously displayed on a single slide. Compared with existing EU staining methods 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 improving the utilization value of sample slides, especially rare samples.

[0039] 2. In view of the problems that existing EU staining is limited to the cellular level, poor tissue penetration (EU requires the assistance of a permeabilizing agent), lack of anatomical location reference markers, and easy fluorescence interference caused by continuous staining, this invention proposes a slc12a3 / EU / LTL triple marker sequential staining method, which overcomes the above obstacles and fills the gap in the technology of tissue localization detection of nascent RNA synthesis level.

[0040] 3. The multiplex immunofluorescence staining method for locating and detecting the level of nascent RNA synthesis proposed in this invention allows for imaging of stained samples using an ultra-high resolution laser confocal microscope. The resulting images 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 level of nascent RNA synthesis. The reagents in this kit can be used for staining and localizing the level of nascent RNA synthesis in samples. In particular, LTL, anti-slc12a3 polyclonal antibody, etc. are used for the first time in the detection of nascent RNA, which greatly improves the accuracy and efficiency of detection, thereby more accurately reflecting the disease state.

[0042] 5. The multiplex immunofluorescence detection method for localizing and detecting the level of neonatal RNA synthesis proposed in this invention combines the azide reaction with localization markers, explores the optimal detection method, and clearly provides experimental steps for detecting neonatal RNA in animal tissues. It realizes how to achieve in situ, multiplex, and quantitative localization analysis of neonatal RNA synthesis in organ tissues, breaks through the limitation of existing technologies that are limited to cell-level detection, solves the problem of spatial heterogeneity resolution in tissue microenvironment, and provides a new approach for efficient and rapid detection of neonatal RNA at the tissue level. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the process for locating and detecting the level of nascent RNA synthesis according to the present invention.

[0044] Figure 2 This is a schematic diagram of the multiplex immunofluorescence staining process in this invention.

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

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

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

[0048] Figure 6 This is a statistical analysis chart comparing simple EU staining and multiplex immunofluorescence staining for samples 1-10.

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

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

[0051] Figure 9 The images show the staining comparison of normal mouse kidney tissue and kidney tissue samples from mice with kidney fibrosis, as shown in Figures 1-3.

[0052] Figure 10 The images show a comparison of staining normal mouse kidney tissue and kidney tissue samples from mice with kidney fibrosis, as shown in images 4-6.

[0053] Figure 11 A statistical analysis of staining comparisons between normal mouse kidney tissue and kidney tissue samples from mice with kidney fibrosis. Detailed Implementation

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

[0055] The overall technical concept of this invention is as follows: Fluorescence resonance energy transfer (FRET) refers to the physical phenomenon of energy being transferred from one excited fluorophore to another. The former is called the donor, and the latter is called the acceptor. When two fluorescent dyes are sufficiently close, FRET will occur. The excited-state donor transfers some energy to the acceptor in a non-radiative manner, causing the acceptor to be excited and emit fluorescence, while its own fluorescence is quenched. To avoid FRET leading to poor staining results, this invention optimizes the concentrations (i.e., dilution factors, see Table 1) of the slc12a3 antibody and LTL stock solution, the staining sequence of the marker, and the marker-fluorescent dye pairing relationship. This determines the optimal antibody concentration, staining sequence, and marker-fluorescent dye pairing, effectively eliminating staining problems caused by FRET.

[0056] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0057] Example 1

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

[0059] (1) Preparation of the sample to be stained: Figure 1 This is a schematic diagram of the process for locating and detecting the level of nascent RNA synthesis in this invention, as shown below. Figure 1 The sample preparation was carried out as shown. All samples were injected intraperitoneally with EU solution 6 hours before sampling, and all were paraffin-embedded tissues fixed with 4% paraformaldehyde. The paraffin tissues were sectioned with a thickness of 3-5 μm and mounted on positively charged glass slides. All samples were assessed for tissue damage by H&E staining. The prepared sections were baked in a 70℃ oven for 2 hours to obtain the stained samples.

[0060] (2) Reagent preparation: The primary antibody for the distal renal tubule marker slc12a3 was diluted with antibody diluent to obtain the primary antibody working solution; the secondary antibody for the distal renal tubule marker slc12a3 was diluted with antibody diluent to obtain the secondary antibody working solution; EU staining working solution was prepared with enzyme-free water, BTTAA, CuSO4, Cy3 azide fluorescent dye, and sodium ascorbate; the LTL stock solution for the proximal renal tubule marker was diluted with antibody diluent to obtain the LTL staining working solution; the main reagent information in this embodiment is shown in Table 1.

[0061] (3) The smears to be stained were first stained with the distal tubule marker Slc12a3. The staining procedure included: dewaxing, antigen retrieval, neutralization, tissue permeation, blocking, primary antibody incubation, secondary antibody incubation, and PBS elution. Next, EU staining was performed, with the following steps: EU staining working solution incubation and permeabilizer elution. Finally, the proximal tubule marker LTL was stained, with the following steps: LTL staining working solution incubation and PBS elution. The staining process is as follows: Figure 2 As shown in Table 2, the pairing relationship between the marker and the fluorescent dye and the dyeing order are shown in Table 3, and the dyed sample is obtained.

[0062] (4) After the staining procedure is completed, the stained sample is taken out, Dapi anti-fluorescence quenching mounting medium is added for staining, and the sample is sealed with a coverslip to obtain the sample to be tested.

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

[0064] Table 1. Main Reagent Information

[0065]

[0066] Table 2. Pairing relationship between markers and fluorescent dye excitation wavelengths and staining order

[0067]

[0068] Table 3 Staining Procedure

[0069]

[0070] Note: The antigen retrieval solution is a modified sodium citrate antigen retrieval solution; the neutralization solution is a 2 mg / ml glycine solution; the tissue permeation agent is a 0.5% Triton X-100 solution; the blocking solution is a 5% BSA solution; and the washing solution is a PBS solution.

[0071] Example 2

[0072] The purpose of this embodiment is to compare and verify the effectiveness of the multiplex immunofluorescence detection method of the present invention with that of the simple EU staining method.

[0073] Ten mouse kidney tissue samples were selected (mice were purchased from Ratliffe, male C57BL / 6JNifdc mice). For each sample, tissue sections were prepared according to step (1) of Example 1. One section was used for multiplex immunofluorescence staining confocal microscopy imaging, and another was used for simple EU staining imaging using conventional fluorescence microscopy. The multiplex immunofluorescence staining and analysis methods are as described in Example 1; the specific steps for simple EU staining are shown in Table 4. Images of multiplex immunofluorescence staining and simple EU staining are shown in Table 4. Figures 3-5 The staining statistics are shown in Table 5. (See Table 5 and...) Figure 6 As shown, multiplex immunofluorescence staining is significantly more effective than simple EU staining, and multiplex immunofluorescence staining can also more intuitively locate and observe the level of nascent RNA synthesis based on images.

[0074] Table 4 Simple EU staining procedure

[0075]

[0076] Table 5. Statistical analysis of multiplex immunofluorescence staining and simple EU staining results

[0077]

[0078] Example 3

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

[0080] Six mouse kidney tissue samples were selected (mice were purchased from Schulich, male C57BL / 6JNifdc mice). For each sample, three serial tissue sections were prepared according to step (1) of Example 1. One section was used for multiplex immunofluorescence staining confocal microscopy imaging, and two sections were used for dual immunofluorescence staining confocal microscopy imaging. One section 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 multiplex immunofluorescence staining and analysis methods were as described in Example 1. Specific steps for the dual immunofluorescence staining method are shown in Tables 6 and 7. Images of the multiplex and dual immunofluorescence staining are shown in [Table 6]. Figure 7 and Figure 8 .like Figure 7 and Figure 8As shown, multiple immunofluorescence staining can simultaneously display the synthesis level of newly generated RNA in both the proximal and distal tubules compared to dual immunofluorescence staining, significantly improving the sample utilization value of dual immunofluorescence staining, while also saving on staining reagents and reducing staining costs.

[0081] Table 6. Dual Immunofluorescence Staining Procedure (LTL and EU Staining)

[0082]

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

[0084]

[0085] Example 4

[0086] The purpose of this embodiment is to demonstrate the application of the multiplex immunofluorescence detection method of the present invention in a renal fibrosis disease model, namely a unilateral ureteral ligation model.

[0087] Kidney tissue samples were selected from 6 normal control mice and 6 mice with renal fibrosis (mice were purchased from Shulaibao, male C57BL / 6JNifdc mice; a unilateral ureteral ligation mouse model was constructed: after anesthesia, the abdominal skin and muscle layers were cut layer by layer along the midline of the abdomen. The left kidney of the mouse was exposed, the left ureter was located, and the ureter was ligated at two points with 5-0 suture. The abdominal organs were returned to the ureter, and the abdominal muscle and skin tissue were sutured layer by layer with 5-0 suture. The abdominal skin wound was disinfected with iodine). For each sample, tissue sections were prepared according to step (1) of Example 1 for multiplex immunofluorescence staining. The staining method was the same as in Example 1, and the staining results are shown in Example 1. Figure 9-10 The statistical analysis results are shown in [the table below]. Figure 11 The experimental results show that the detection kit provided by this invention, when applied to the detection of renal fibrosis, not only allows for direct observation of a significant decrease in the level of newly synthesized RNA in the proximal tubules, but statistical analysis also shows the same significant results. This suggests that the multiplex immunofluorescence staining kit provided by this invention for the localization and detection of newly synthesized RNA levels has promising applications in the preparation of products for the detection of renal fibrosis.

[0088] In summary, the multiplex immunofluorescence staining method of this invention for the localization and detection of nascent RNA synthesis levels achieves both staining and localization detection of nascent RNA synthesis levels in test samples, filling the gap in tissue localization detection technology for nascent RNA synthesis levels. By using multiplex immunofluorescence staining to detect nascent RNA synthesis levels, the expression of nascent RNA in different sites can be simultaneously displayed on a single slide, improving the utilization value and efficiency of sample slides. This method enables simpler, more efficient, and more accurate detection of tissue nascent RNA synthesis levels, thereby more accurately reflecting disease status.

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

Claims

1. A multiplex immunofluorescence staining method, characterized in that, Includes the following steps: S1. Prepare the sample slides to be stained; S2. Prepare slc12a3 staining working solution, EU staining working solution and LTL staining working solution respectively. The slc12a3 staining working solution includes slc12a3 primary antibody working solution and slc12a3 secondary antibody working solution. The slc12a3 primary antibody working solution and the slc12a3 secondary antibody working solution were obtained by diluting the slc12a3 primary antibody and the fluorescent secondary antibody at a ratio of 1:280-320, respectively; each milliliter of the EU staining working solution contained 930-940 μL of enzyme-free water, 45-55 μL of 10 mM BTTAA, 8-12 μL of 2 mM CuSO4, 2.5-3.5 μL of 0.1% Cy3 azide fluorescent dye, and 8-10 mg of sodium ascorbate; the LTL staining working solution was obtained by diluting the LTL stock solution at a ratio of 1:80-120. The primary antibody for slc12a3 includes a rabbit-derived polyclonal antibody; the fluorescent secondary antibody for 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. S3. The stained specimens are stained sequentially with slc12a3 staining, EU staining and LTL staining to obtain stained specimens. S4. Restain with fluorescent quenching mounting medium and mount to obtain the sample to be tested.

2. The multiplex immunofluorescence staining method according to claim 1, characterized in that, The preparation method of the stained sample section in step S1 includes: fixing and embedding the tissue treated with EU solution, then cutting it into sections with a thickness of 3-5 μm, and then baking it at 65-75℃ for 1.5-2.5 h.

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

4. The multiplex immunofluorescence staining method according to claim 1, characterized in that, The fluorescence quenching mounting medium mentioned in step S4 includes a mounting medium containing Dapi anti-fluorescence quenching agent.

5. A multiplex immunofluorescence detection method, characterized in that, The method includes the step of using the multiplex immunofluorescence staining method according to any one of claims 1-4, and further includes the steps of microscopic imaging, image analysis, and obtaining analytical data on the level of nascent RNA synthesis.

6. The application of the multiplex immunofluorescence staining method according to any one of claims 1-4 in the preparation of products for detecting the level of nascent RNA synthesis.

7. A product for detecting the level of nascent RNA synthesis, characterized in that, The product includes a kit containing the reagents used in any of the multiplex immunofluorescence staining methods of claims 1-4.

8. The use of the multiplex immunofluorescence staining method according to any one of claims 1-4 and / or the product according to claim 7 in the preparation of a kidney fibrosis detection product.

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