A method for immunofluorescence staining of cells that can amplify signals

Abstract

The present invention discloses a method for cell immunofluorescence staining capable of amplifying signals, belonging to the field of biotechnology. In the present invention, the aptamer HCR primer is anchored to the cell surface target, and an HCR reaction is carried out to extend a fluorescent HCR product chain, thereby realizing cell immunofluorescence staining. Compared with the traditional antibody staining technology, the fluorescence signal intensity of the present invention is increased by 1.5 times.

Description

Technical Field

[0001] The present invention relates to a method for immunofluorescent staining of cells capable of amplifying signals, belonging to the field of biotechnology. Background Art

[0002] Immunofluorescent staining is one of the commonly used methods for detecting the expression of cell surface proteins. Its principle is usually to use an antibody conjugated with a fluorescent molecule to specifically recognize and bind to the target, enriching the fluorescent molecule on the surface of the target, thereby achieving fluorescent staining of the target protein. However, for low-abundance targets, simply using one antibody modified with one fluorescent molecule for targeted recognition cannot show sufficient brightness for observation and analysis. Therefore, signal amplification technology is indispensable in the process of immunofluorescent staining to enhance the fluorescent signal and improve the detection sensitivity.

[0003] The most widely used method in immunofluorescent staining is to use a primary antibody to recognize the antigen target, and then use a secondary antibody modified with a fluorescent molecule to target and recognize the primary antibody, thereby achieving visualization of the target. (Han, K.N., Li, C.A. & Seong, G.H. Annu. Rev. Anal. Chem. 6, 119–141 (2013)) However, each primary antibody can only bind 1 - 2 secondary antibodies, and each secondary antibody can only be modified with 2 - 3 fluorescent molecules. This very basic level of signal amplification restricts the intensity of the output signal and can only meet the detection requirements for medium- and high-abundance targets. At the same time, antibodies are essentially proteins, but proteins are easily denatured by factors such as temperature and pH, which puts high requirements on the storage and transportation conditions of the reaction reagents. However, due to its simple operation, traditional antibody staining still remains the most practical staining method. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for immunofluorescent staining of cells capable of amplifying signals, which can enhance the signal intensity after fluorescent staining of cell surface protein targets, and improve the brightness and detection sensitivity of low-abundance targets.

[0005] The present invention combines aptamers and HCR, anchors the HCR primers on the target using aptamers capable of specifically recognizing the target, and then adds hairpin probes H1 and H2 modified with fluorescent molecules to in-situ extend a long HCR chain labeled with a large number of fluorescent molecules, thereby constructing a brand-new signal amplification system.

[0006] An aptamer is a single-stranded DNA fragment that binds to a target with high affinity and specificity using a specific secondary structure. Its affinity is similar to that of an antibody, both with KD = 10 -9Around

[0007] The technical solution for achieving the above object is as follows:

[0008] The present invention provides an aptamer-HCR primer complex, and its nucleotide sequence is shown in SEQ ID NO.1.

[0009] The present invention also provides a cell immunofluorescence staining kit, which includes an aptamer-HCR primer complex, hairpin probe H1 and hairpin probe H2.

[0010] Further, in the above technical solution, the hairpin probe H1 is a nucleotide sequence shown in SEQ ID NO.2 with a fluorescent group modified at the 3' end.

[0011] Further, in the above technical solution, the hairpin probe H2 is a nucleotide sequence shown in SEQ ID NO.3 with a fluorescent group modified at the 5' end.

[0012] Further, in the above technical solution, the fluorescent group includes Alexa Fluor 488.

[0013] The present invention also provides a cell immunofluorescence staining method capable of amplifying signals. Anchor the aptamer-HCR primer complex on the cell surface, add hairpin probe H1 and hairpin probe H2, perform an in-situ HCR reaction, and extend a fluorescent HCR product chain to achieve cell immunofluorescence staining.

[0014] Further, in the above technical solution, it includes the following steps:

[0015] (1) Heat the aptamer-HCR primer complex solution at 85-95 °C for 90-95 s, then cool it to room temperature for 10-20 min, and dilute the cooled aptamer-HCR primer complex solution in a hybridization buffer to a final concentration of 0.25-0.5 uM;

[0016] (2) Add the aptamer-HCR primer complex solution obtained in step (1) to the cells and incubate at room temperature for 1-2 h, and wash at least three times with 2xSSC;

[0017] ​(3) Heat the hairpin probe H1 and hairpin probe H2 solutions at 85 - 95 °C for 90 - 95 s, then cool them to room temperature for 10 - 20 min. Dilute the cooled H1 and H2 solutions in the hybridization buffer to a final concentration of 0.25 - 0.5 μM;

[0018] (4) Add the H1 solution and H2 solution obtained in step (3) to the cells and incubate at room temperature for 3 - 12 h. Rinse with 2×SSC at least three times to complete the immunofluorescence staining of the cells.

[0019] Further, in the above technical solution, the cells include cancer cells.

[0020] Further, in the above technical solution, the cancer cells include triple-negative breast cancer cells and non-small cell lung cancer cells.

[0021] Advantages of the Invention

[0022] 1. The present invention improves the fluorescence signal intensity by 1.5 times compared with traditional antibody staining, enhancing the brightness of low-abundance targets and the detection sensitivity.

[0023] 2. The reagents used in the present invention have strong stability and can be transported and stored at room temperature. Description of the Drawings

[0024] Figure 1 This is a fluorescence microscope photograph of the staining of the PD-L1 target of triple-negative breast cancer cells (MDA-MB-231) using HCR signal amplification immunofluorescence staining in Example 1 of the present invention. The scale bar is 20 micrometers.

[0025] Figure 2 This is a fluorescence microscope photograph of the staining of the PD-L1 target of human non-small cell lung cancer cells (HOP-92) using HCR signal amplification immunofluorescence staining in Example 2 of the present invention. The scale bar is 200 micrometers.

[0026] Figure 3 a is a fluorescence microscope photograph of the staining of the PD-L1 target of MDA-MB-231 cells using traditional antibody staining; Figure 3 b is a fluorescence microscope photograph of the staining of the PD-L1 target of MDA-MB-231 cells using HCR signal amplification immunofluorescence staining; Figure 3 c is the statistical chart of the fluorescence signal intensity. The scale bar is 200 micrometers. Detailed Embodiments

[0027] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way.

[0028] Example 1

[0029] In this example, triple-negative breast cancer cells (MDA-MB-231) purchased from the Cell Bank of the Chinese Academy of Sciences were used as a tumor cell model to further elaborate and verify the HCR signal amplification immunofluorescence staining method of the present invention, including the following steps:

[0030] 1. Prepare the tumor cell sample to be tested:

[0031] Take the tumor cell suspension and count it with a cell counter. After calculating its concentration, aspirate a certain amount of the above cell suspension and dilute the suspension to 1×10 4 cells / mL with DMEM cell culture medium, inject it into a 24-well plate, and statically incubate it in a cell incubator for 12 h.

[0032] 2. Prepare the hybridization buffer:

[0033] 0.1% Tween 20, 10% dextran sulfate, 2x SSC buffer, 1x Denhardt's solution, and make up with double-distilled water.

[0034] 3. HCR signal amplification immunofluorescence staining:

[0035] 1) Cell fixation

[0036] Discard the culture medium in the 24-well plate and rinse it three times with 1xPBS. Add 400 μL of 4% paraformaldehyde to fix for 20 min, and rinse it three times with 1xPBS.

[0037] 2) Cell blocking

[0038] Add 400 μL of 3% skim milk powder to block for 1 h, and rinse it three times with 1xPBS.

[0039] 3) HCR reaction

[0040] Heat the aptamer-HCR primer complex solution (SEQ ID NO.1) at 95 °C for 90 s, then cool it at room temperature for 20 min. Dilute the cooled aptamer-HCR primer complex solution in the hybridization buffer to a final concentration of 0.5 μM. Add 400 μL of the diluted aptamer-HCR primer complex solution to the cells fixed in the 24-well plate and incubate at room temperature for 2 h, and rinse it three times with 2x SSC.

[0041] Heat the hairpin probe H1 (SEQ ID NO.2) and hairpin probe H2 (SEQ ID NO.3) solutions at 95 °C for 90 s, then cool them at room temperature for 20 min. Dilute the cooled H1 and H2 solutions in the hybridization buffer to a final concentration of 0.5 μM. Add 200 μL of the diluted H1 solution and 200 μL of the diluted H2 solution to the cells fixed in the 24-well plate and incubate at room temperature for 12 h, and rinse it three times with 2×SSC.

[0042] 5. DAPI nuclear staining:

[0043] Add 400 μl of DAPI solution diluted 1:100 and stain for 20 min, then rinse three times with 1x PBS.

[0044] Figure 1 The experimental results show that the HCR signal amplification immunofluorescence staining method can stain the protein targets on the surface of tumor cells.

[0045] Example 2

[0046] In this example, human non-small cell lung cancer cells (HOP-92) purchased from the Cell Bank of the Chinese Academy of Sciences were used as the tumor cell model to further elaborate and verify the RCA-HCR signal amplification immunofluorescence staining method of the present invention, including the following steps:

[0047] 1. Prepare the tumor cell sample to be tested:

[0048] The method is the same as that in Example 1.

[0049] 2. Prepare the hybridization buffer:

[0050] The method is the same as that in Example 1.

[0051] 3. HCR signal amplification immunofluorescence staining:

[0052] 1) Cell fixation

[0053] The method is the same as that in Example 1.

[0054] 2) Cell blocking

[0055] The method is the same as that in Example 1.

[0056] 3) HCR reaction

[0057] Heat the aptamer-HCR primer complex solution (SEQ ID NO.1) at 95 °C for 90 s, then cool it at room temperature for 20 min. Dilute the cooled aptamer-HCR primer complex solution in the hybridization buffer to a final concentration of 0.25 μM. Add 400 μL of the diluted aptamer-HCR primer complex solution to the cells fixed in the 24-well plate and incubate at room temperature for 1 h, then rinse three times with 2x SSC.

[0058] Heat the solutions of hairpin probe H1 (SEQ ID NO.2) and hairpin probe H2 (SEQ ID NO.3) at 95 °C for 90 s, then cool them at room temperature for 20 min. Dilute the cooled H1 and H2 solutions in hybridization buffer to a final concentration of 0.25 uM. Add 200 uL of the diluted H1 solution and 200 uL of the diluted H2 solution to the cells fixed in a 24-well plate and incubate at room temperature for 3 h, then rinse three times with 2×SSC.

[0059] 5. DAPI nuclear staining:

[0060] The method is the same as that in Example 1.

[0061] Figure 2 The experimental results show that HCR signal amplification immunofluorescence staining can stain protein targets of different types of tumor cells by changing the aptamer sequence.

[0062] Comparative Example 1

[0063] 1. Prepare tumor cell samples to be tested:

[0064] The method is the same as that in Example 1.

[0065] 2. Antibody staining

[0066] 1) Cell fixation

[0067] Discard the culture medium in the 24-well plate, and rinse three times with 1xPBS. Add 400 ul of 4% paraformaldehyde to fix for 20 min, and then rinse three times with 1xPBS.

[0068] 2) Cell blocking

[0069] Add 400 ul of 3% skim milk powder to block for 1 h, and then rinse three times with 1xPBS.

[0070] 3) Add 400 ul of PD-L1 rabbit primary antibody diluted 1:300, incubate at 37 °C for 1 h, and then rinse three times with 1xPBS. Add 400 ul of goat anti-rabbit secondary antibody diluted 1:300, incubate at 4 °C for 12 h, and then rinse three times with 1xPBS.

[0071] 4) DAPI staining

[0072] Add 400 ul of DAPI solution diluted 1:100 to stain for 20 min, and then rinse three times with 1xPBS.

[0073] Figure 3 a is the fluorescence microscope photo of traditional antibody staining for PD-L1 target staining of MDA-MB-231 cells. Figure 3b is the fluorescence microscope photo of PD-L1 target staining of MDA-MB-231 cells using HCR signal amplification immunofluorescence staining. Figure 3 c is the statistical result of fluorescence signal intensity. The scale bar is 200 microns. Figure 3 The experimental results show that RCA-HCR signal amplification immunofluorescence staining can effectively enhance the fluorescence signal of tumor cells, and the fluorescence signal intensity is increased by 1.5 times compared with traditional antibody staining.

[0074] Table 1. Oligonucleotide sequences and modifications

[0075]

[0076] The above embodiments are only used for exemplifying and explaining the present invention, and are not intended to limit the present invention within the scope of the described embodiments. In addition, those skilled in the art can understand that the present invention is not limited to the above embodiments, and more variations and modifications can be made according to the teachings of the present invention, and these variations and modifications all fall within the scope of the present invention claimed. SEQUENCE LISTING <110> Dalian University of Technology <120> A method for cell immunofluorescence staining with signal amplification <130> 2022 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 86 <212> DNA <213> Artificial Sequence <400> 1 acgggccaca tcaactcatt gatagacaat gcgtccactg cccgtatata gcattctttc 60 ttgaggaggg cagcaaacgg gaagag 86 <210> 2 <211> 72 <212> DNA <213> Artificial Sequence <400> 2 cgtaaaggaa gactcttccc gtttgctgcc ctcctcgcat tctttcttga ggagggcagc 60 aaacggggaag ag 72 <210> 3 <211> 72 <212> DNA <213>人工序列(Artificial Sequence) <400> 3 gaggagggca gcaaacgggga agagtcttcc tttacgctct tcccgtttgc tgccctcctc 60 aagaaagaat gc 72

Claims

1. An aptamer-HCR primer complex, characterized in that: The nucleotide sequence is as shown in SEQ ID NO.

1.

2. A cell immunofluorescence staining kit, characterized in that: Comprising the aptamer-HCR primer complex, hairpin probe H1 and hairpin probe H2 as described in claim 1; The hairpin probe H1 is a nucleotide sequence shown in SEQ ID NO.2 with a fluorophore modified at the 3'-end; The hairpin probe H2 is a nucleotide sequence shown in SEQ ID NO.3 with a fluorophore modified at the 5'-end.

3. The kit according to claim 2, wherein: The fluorophore includes Alexa Fluor 488.

4. A method for immunofluorescent staining of cells that can amplify signals, characterized in that: Anchoring the aptamer-HCR primer complex to the cell surface, adding hairpin probe H1 and hairpin probe H2, performing an in-situ HCR reaction to extend a fluorescent HCR product strand, and achieving cell immunofluorescence staining; the nucleotide sequence of the aptamer-HCR primer complex is as shown in SEQ ID NO.1; the hairpin probe H1 is a nucleotide sequence shown in SEQ ID NO.2 with a fluorophore modified at the 3'-end; the hairpin probe H2 is a nucleotide sequence shown in SEQ ID NO.3 with a fluorophore modified at the 5'-end.

5. The method according to claim 4, characterized in that: Comprising the following steps: (1) Heating the aptamer-HCR primer complex solution at 85 - 95 °C for 90 - 95 s, then cooling it to room temperature for 10 - 20 min, and diluting the cooled aptamer-HCR primer complex solution in hybridization buffer to a final concentration of 0.25 - 0.5 uM; (2) Adding the aptamer-HCR primer complex solution obtained in step (1) to the cells and incubating at room temperature for 1 - 2 h, then rinsing with 2x SSC at least three times; (3) Heating the hairpin probe H1 and hairpin probe H2 solutions at 85 - 95 °C for 90 - 95 s, then cooling them to room temperature for 10 - 20 min, and diluting the cooled H1 and H2 solutions in hybridization buffer to a final concentration of 0.25 - 0.5 uM; (4) Adding the H1 solution and H2 solution obtained in step (3) to the cells and incubating at room temperature for 3 - 12 h, then rinsing with 2×SSC at least three times to complete cell immunofluorescence staining.

6. The method according to claim 5, wherein: The cells include cancer cells.

7. The method according to claim 6, characterized in that: The cancer cells include triple-negative breast cancer cells and non-small cell lung cancer cells.

Images