A method for in situ protein immunofluorescence cyclic staining and data analysis

Abstract

The application provides a method for in-situ protein immunofluorescence cycle staining and data analysis, and belongs to the field of biological detection. The specific steps of the method are as follows: S1. preparing a sample tissue section; S2. fluorescence staining; S3. elution: elution is performed using an Aibixin abs994 eluent; S4. cycle staining: after elution is completed, cycle staining is performed by repeating steps S2-S3 according to the detected protein; a fluorescence staining image is obtained after each staining is completed; S5. cell nucleus image recognition; S7. multi-round image registration; S8. antibody-positive cell screening and connectivity segmentation based on overlapping areas: different cell identities are divided based on the overlapping areas with specific cell nuclei, the surrounding relationship and the repulsion relationship between different antibodies, so that in-situ detection of multiple proteins can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a method for tissue in-situ protein immunofluorescence cycle staining and data analysis. BACKGROUND

[0002] Tissue in-situ protein staining technology plays an important role in basic research and clinical diagnosis. This method uses specific primary antibodies to recognize target proteins, and through the binding of secondary antibodies labeled with fluorescent groups or enzymes, the expression of target proteins is displayed. Traditional tissue in-situ protein staining technology can only detect up to 4 proteins on the same tissue section, significantly limiting the application of this technology. In order to break this limitation, multi-round immunofluorescence staining technology has been developed for in-situ detection of multiple protein expression. Most of the current multi-round immunofluorescence staining technology relies on the quenching of fluorescent groups and the specific modification of antibodies, which is complicated to operate, expensive in reagents, and limited to special equipment, which is not conducive to popularization in most laboratories. Therefore, there is currently a need to develop a simple method suitable for detecting multiple protein expression.

[0003] Patent No. CN117887820A, entitled "A method for simultaneously in-situ fluorescent detection of human RNA, DNA and protein" discloses that the TSA signal amplification system can preserve RNA-FISH and protein immunofluorescence signals in the high temperature and strong acid environment during the DNA-FISH experiment, thereby achieving simultaneous in-situ detection of RNA, DNA and protein signals in cells and tissues. Specifically, through the catalysis of horseradish peroxidase (HRP), TSA fluorescent compounds can be bound to tyrosine residues near the target, and stable fluorescent compounds resistant to heat and acid are generated. Through the DNA-FISH experiment, the DNA signal is finally obtained. However, this technology can only detect one RNA, one DNA and one protein at the same time, which has a very significant limitation.

[0004] Patent No. CN118311267A, entitled "Multi-protein and nucleic acid detection method using color combination and sequential fluorescence in-situ hybridization (CCS-FISH) assisted by rolling circle amplification (RCA)" discloses that oligonucleotide-labeled antibodies are used, and after the antibodies recognize the target protein, the oligonucleotide is amplified by rolling circle amplification. Since the oligonucleotide contains a binding site for a fluorescent probe, the expression of the protein can be indicated by the fluorescent color. However, this method requires oligonucleotide labeling of the protein, which inevitably destroys the structure of the antibody and affects the specificity of the antibody, in addition, rolling circle amplification can cause signal distortion, which is not completely consistent with the in-situ protein signal.

[0005] Therefore, there is an urgent need for a method for accurately and simply detecting multiple proteins in-situ. SUMMARY

[0006] Therefore, the present application aims to provide a method for in-situ protein immunofluorescence cyclic staining and data analysis, which can realize in-situ detection of multiple proteins.

[0007] In order to achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a method for in-situ protein immunofluorescence cyclic staining and data analysis, comprising the following steps:

[0009] S1. Preparing a sample tissue section;

[0010] S2. Fluorescent staining;

[0011] S3. Elution:

[0012] After heating the antibody eluent, it is added dropwise to the fluorescently stained tissue section on a shaking table at room temperature. Then the antibody eluent is aspirated, and the antibody eluent is added again. The slide is sealed and treated in a water bath. Then the antibody eluent is aspirated, and the slide is washed with phosphate buffer;

[0013] S4. Cyclic staining:

[0014] After elution is complete, steps S2-S3 are repeated for cyclic staining according to the target protein to be detected. After each staining is complete, a fluorescent staining image is obtained;

[0015] S5. Nucleus image recognition:

[0016] After each cycle of staining, after the last staining elution is complete, the tissue section is stained with DAPI to stain the nucleus. After maximum density projection in the z-axis direction, the pre-trained model of the Cellpose method is used to segment the nucleus image in the 2D layer to obtain a cell mask and number, and the nucleus area is used for quality control screening to remove incorrectly divided nuclei;

[0017] S6. Differentiation and enhancement of antibody images:

[0018] The images of different antibodies obtained in the cyclic staining are denoised to facilitate subsequent division of antibody positive regions;

[0019] S7. Multi-round image registration:

[0020] Multi-round image registration uses a feature-based registration method to calculate the transformation relationship between rounds based on the DAPI channel image of each round, and migrates and applies it in the form of a homography matrix to the remaining antibody channels in the same round to achieve spatial registration of antibody signals;

[0021] S8. Antibody-positive cell screening and connectivity segmentation based on overlapping area:

[0022] Different cell identities are divided based on the overlapping area, surrounding relationship with specific cell nucleus and the repulsion relationship between different antibodies. The cell identity is determined by the relative relationship between the antibodies and the cell nucleus in the image, the overlapping area of the effective signal (after connectivity and gray scale screening) in the given area (in the nucleus or around the nucleus) and the pixel intensity of the overlapping area to give the likelihood probability, and the final cell identity is given by comparing the likelihood probability under different antibody conditions.

[0023] As preferred, the sample preparation in S1 is one of paraffin section, frozen section and adherent cell.

[0024] Further preferably, the specific method for sample preparation is:

[0025] (1) Tissue cryopreservation

[0026] Fresh tissue is fixed with 4% PFA at 4°C overnight on a shaker;

[0027] Rinse the tissue with phosphate buffer solution twice, and dehydrate with 30% sucrose;

[0028] After the tissue sinks to the bottom of the sucrose solution, transfer the tissue to OCT and freeze it on dry ice;

[0029] After the quick freezing is completed, store the tissue in a -80°C refrigerator;

[0030] (2) Slide coating

[0031] Soak the slide in 1M HCL solution at 40 rpm on a shaker for 1 h, rinse with water twice, then soak in 1M NaOH at 40 rpm on a shaker for 1 h, and rinse with water twice;

[0032] Soak the slide in 1% silane solution at pH 3.5 at 40 rpm on a shaker for 1 h, and rinse with ethanol three times;

[0033] Soak the slide in ethanol three times, each time for 15 min, and then place it in a 90°C oven for 30 min;

[0034] (3) Sectioning

[0035] Place the OCT cryopreserved tissue on the sample head of the microtome;

[0036] Cut a tissue section with a thickness of 18 μm;

[0037] Carefully spread the tissue section with a brush pen;

[0038] Place the coated slide close to the section to adhere it to the slide.

[0039] As preferred, the specific step S2 is:

[0040] Put the glass slide with tissue section into 60℃ oven for 1h; then fix the glass slide in 2% PFA for 20min, and wash in PBS for 3 times, 10min each time;

[0041] Then add appropriate amount of blocking solution on the tissue section and treat for 20min; add primary antibody on the tissue section, cover with sealing film, and shake overnight at 4℃; the blocking solution is PBS containing 5% donkey serum and 0.3% Triton-X100;

[0042] Then soak in PBS for 3 times, 10min each time, and then add corresponding secondary antibody on the tissue section, shake for 2h at room temperature;

[0043] Then soak in PBS for 2 times, 10min each time, and then add 1μg / ml DAPI on the tissue section, treat for 10min, and then soak in PBS for 10min again; use 65% glycerol solution for mounting;

[0044] Finally obtain the fluorescent staining image.

[0045] As preferred, in S3:

[0046] The eluent is Aibixin abs994;

[0047] The heating temperature of the eluent is 25-37℃;

[0048] The shaking time is 5-10min;

[0049] The sealing method uses plastic bag;

[0050] The water bath treatment temperature is 37-50℃, and the time is 30-60min.

[0051] As preferred, in S2 or S4, the method for obtaining the fluorescent staining image uses laser confocal microscope or ordinary fluorescent microscope for photographing.

[0052] As preferred, in S6, the noise reduction method is:

[0053] Combination of gray scale histogram equalization, image morphological operation and median filter denoising method is used to operate on pixels and patterns, so as to realize the strengthening of local features of antibody image.

[0054] As preferred, the image recognition of DAPI uses the pre-trained model of cellpose2.0.

[0055] At least the following beneficial technical effects are contained:

[0056] The present application can realize in-situ detection of multiple proteins by simply increasing the imaging rounds. The complex reagent configuration is greatly simplified, and the cost is reduced while ensuring the accuracy of experimental results. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 Figure 4 is a continuous three-round immunofluorescence imaging diagram of brain tissue sections of 5xFAD mice.

[0058] Figure 2 Figure 5 is a continuous three-round immunofluorescence imaging superimposition diagram of brain tissue sections of 5xFAD mice. DETAILED DESCRIPTION

[0059] The technical solutions of the present application are further described below by means of the accompanying drawings and examples.

[0060] Unless otherwise defined, technical or scientific terms used in the present application should be understood as having the commonly understood meaning in the field of the present application to which the person with ordinary skill in the art belongs.

[0061] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims should be included in the present application, and any reference signs in the claims should not be regarded as limiting the claims involved.

[0062] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be combined appropriately to form other embodiments that those skilled in the art can understand. These other embodiments are also covered by the protection scope of the present application.

[0063] It should also be understood that the specific embodiments described above are only for the purpose of explaining the present application, and the protection scope of the present application is not limited thereto, and any person skilled in the art can make equivalent replacements or changes to the technical solutions and inventive concepts of the present application within the technical scope disclosed by the present application, which should be covered by the protection scope of the present application / invention.

[0064] EMBODIMENTS

[0065] S1. Preparation of sample tissue sections: The brain tissue of 5xFAD mice at 12 months of age was removed after heart perfusion with PBS, fixed with 4% PFA overnight, dehydrated with 30% sucrose, embedded with OCT and frozen on dry ice. The frozen sections were cut in a freezing microtome and then pasted on PDL-coated glass slides.

[0066] S2. Fluorescent staining: The glass slides with tissue sections were baked in an oven at 60°C for 1 hour, fixed in 2% PFA for 20 min, and washed with phosphate buffer for 3 times, 10 min each time. Then 5% donkey serum was blocked for 20 min, and then the CD68, Aβ and Olig2 antibodies were incubated, and the corresponding secondary antibodies were applied, washed with PBS for 3 times, 10 min each time, finally stained with DAPI, washed with PBS once, and photographed. The first round of antibodies was removed by antibody elution solution (Aibio abs994), and the second round (GFAP, Iba1, AldC) and the third round (Sox1, Sox10, Sox9) antibodies were incubated in the same way, and after the end of each round, the antibody elution solution (Aibio abs994) was used for elution, and photographed respectively.

[0067] Finally, the DAPI signal of each round was used for registration, and the staining signals of all proteins were superimposed into the same picture.

[0068] Figure 1 For the continuous 3 rounds of in situ protein staining of 5xFAD mouse brain slices, the shooting part is the hippocampal region of the mouse.

[0069] Figure 2 The superimposed image of the obtained three rounds of protein staining signals.

Claims

1. A method for in situ immunofluorescence cyclic staining and data analysis of proteins in tissues, characterized in that, Includes the following steps: S1. Prepare tissue sections for the sample; S2. Fluorescent staining; S3. Washing: After heating the antibody elution buffer, add it dropwise onto the fluorescently stained tissue section and shake at room temperature; then aspirate the antibody elution buffer, add the antibody elution buffer again, seal the slide, and treat it in a water bath; then aspirate the antibody elution buffer and wash with phosphate buffer. S4. Cyclic staining: After elution, steps S2-S3 are repeated for cyclic staining according to the target protein to be detected; fluorescent staining images are obtained after each staining. S5. Cell nucleus image recognition: After each cycle of staining, once the last staining and elution is completed, the tissue sections are stained with DAPI to stain the cell nuclei. After maximum density projection in the z-axis direction, the pre-trained model of the Cellpose method is used to segment the cell nucleus image in 2D, obtain cell masks and numbers, and use the cell nucleus area for quality control screening to remove cell nuclei with incorrect boundary division. S6. Differential enhancement of antibody images: Denoising the images of different antibodies obtained from cyclic staining facilitates the subsequent segmentation of antibody-positive regions. S7. Multi-round image registration: Multi-round image registration uses a feature-based registration method, which calculates the transformation relationship between rounds based on the DAPI channel image of each round, and applies it to the remaining antibody channels in the same round in the form of homography matrix to achieve spatial registration of antibody signals. S8. Antibody-positive cell screening and connectivity segmentation based on overlapping area: By analyzing the distribution characteristics of different antibodies, and based on the overlap area with specific cell nuclei, the surrounding relationship, and the repulsion relationship between different antibodies, different cell identities are classified.

2. The method according to claim 1, characterized in that, The sample in S1 is prepared as one of the following: paraffin section, frozen section, or adherent cells.

3. The method according to claim 1, characterized in that, The specific steps of S2 are as follows: Place the slides with the tissue sections attached into a 60°C oven and bake for 1 hour; then fix the slides in 2% PFA for 20 minutes, and wash them 3 times with phosphate buffer, each time for 10 minutes. Then, an appropriate amount of blocking solution was added to the tissue section and treated for 20 min; primary antibody was added to the tissue section, covered with sealing film, and incubated overnight at 4°C on a shaker; the blocking solution was phosphate buffer containing 5% donkey serum and 0.3% Triton-X100. Then soak the tissue sections three times with phosphate buffer for 10 minutes each time, and then add the corresponding secondary antibody to the tissue sections and shake at room temperature for 2 hours. Then, soak the tissue sections twice with phosphate buffer for 10 minutes each time. After that, add 1 μg / ml DAPI to the tissue sections, treat for 10 minutes, and then soak again in phosphate buffer for 10 minutes. Mount the sections with 65% glycerol solution. Finally, fluorescently stained images were obtained.

4. The method according to claim 1, characterized in that, In S3: The eluent is Absin ABS994; The heating temperature of the eluent is 25–37°C; The shaking time is 5 to 10 minutes; The sealing method uses a plastic bag; The water bath treatment temperature is 37–50°C, and the time is 30–60 minutes.

5. The method according to claim 1, characterized in that, The fluorescent staining images are obtained in S2 or S4 by taking pictures using a laser confocal microscope or a regular fluorescence microscope.

6. The method according to claim 1, characterized in that, The noise reduction method in S6 is as follows: By combining grayscale histogram equalization, image morphology operations, and median filtering denoising methods to manipulate pixels and patterns, the local features of antibody images can be enhanced.

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