Method for tissue in-situ protein immunofluorescence cycle staining and data analysis

Abstract

The invention provides a method for tissue in-situ protein immunofluorescence circulating staining and data analysis, and belongs to the field of biological detection. The method comprises the following specific steps: S1, preparing a sample tissue slice; s2, carrying out fluorescent staining; s3, elution: carrying out elution by adopting an Elbixt abs994 eluent; s4, circulating dyeing: after the elution is completed, repeating the steps S2 to S3 according to the detected target protein to perform circulating dyeing; obtaining a fluorescent dyeing image after each dyeing is finished; s5, identifying a cell nucleus image; s7, performing multi-round image registration; and S8, antibody positive cell screening and connectivity segmentation based on the overlapping area: through distribution characteristics of different antibodies, based on the overlapping area with a specific cell nucleus, a surrounding relationship and a rejection relationship before different antibodies, different cell identities are divided, and in-situ detection of a plurality of proteins can be realized.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and in particular relates to a method for tissue in situ protein immunofluorescence cycle staining and data analysis. Background Art

[0002] Tissue in situ protein staining technology plays an important role in basic research and clinical diagnosis. This method uses a specific primary antibody to identify the target protein, and binds to the primary antibody through a fluorescent group or enzyme-labeled secondary antibody to display the expression of the target protein. Traditional tissue in situ protein staining technology can only detect up to 4 proteins on the same tissue section, which significantly limits the application of this technology. In order to break through this limitation, multiple rounds of immunofluorescence staining technology have been developed for in situ detection of the expression of multiple proteins. Most of the current multiple rounds of immunofluorescence staining techniques rely on the quenching of fluorescent groups and the specific modification of antibodies. The operation is cumbersome, the reagents are expensive, and they are limited by special equipment, which is not conducive to promotion in most laboratories. Therefore, there is a need to develop a simple method suitable for detecting the expression of multiple proteins.

[0003] Patent number CN117887820A, entitled "A method for simultaneous in situ fluorescence detection of human RNA, DNA, and protein", discloses that the use of the TSA signal amplification system can retain RNA-FISH and protein immunofluorescence signals in the high temperature and strong acid environment during the DNA-FISH experiment, thereby realizing the simultaneous in situ detection of RNA, DNA, and protein signals in cells and tissues. The method is specifically catalyzed by horseradish peroxidase (HRP), and the TSA fluorescent compound can be bound to the tyrosine residues near the target site to generate a heat-resistant and acid-resistant stable fluorescent compound. The DNA signal is finally obtained through the DNA-FISH experiment. However, this technology can only detect one RNA, one DNA, and one protein at the same time, which has very significant limitations.

[0004] Patent number CN118311267A, named "Multiple protein and nucleic acid detection method using rolling circle amplification (RCA) assisted color combination and sequential fluorescence in situ hybridization (CCS-FISH)" Chinese patent discloses the use of oligonucleotides to label antibodies, and after the antibody recognizes the target protein, the oligonucleotides are amplified by rolling circles. Since the oligonucleotide contains binding sites for fluorescent probes, 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 antibody structure and affects the specificity of the antibody. In addition, rolling circle amplification will cause signal distortion and is not completely consistent with the in situ protein signal.

[0005] Therefore, there is an urgent need for an accurate and simple method to detect multiple proteins in situ. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a method for in situ protein immunofluorescence cycle staining and data analysis of tissues, which can realize in situ detection of multiple proteins.

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

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

[0009] S1. Prepare sample tissue sections;

[0010] S2. Fluorescence staining;

[0011] S3. Elution:

[0012] Heat the antibody eluate and drip it onto the fluorescently stained tissue sections and shake them at room temperature; then remove the antibody eluate, drip it again, seal the slide, and treat it in a water bath; then remove the antibody eluate and wash it with phosphate buffer;

[0013] S4. Cycle dyeing:

[0014] After the elution is completed, steps S2 to S3 are repeated according to the detection target protein to perform cyclic staining; after each staining, a fluorescent staining image is obtained;

[0015] S5. Cell nucleus image recognition:

[0016] After each round of staining, after the last staining and washing is completed, the tissue sections are stained with DAPI to stain the cell nuclei. After the 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 the cell mask and number, and use the cell nucleus area for quality control screening to eliminate the cell nuclei with incorrect boundary division;

[0017] S6. Differential enhancement of antibody images:

[0018] The images of different antibodies obtained by cyclic staining were denoised to facilitate the subsequent division of antibody-positive areas;

[0019] S7. Multiple rounds of image registration:

[0020] Multi-round image registration uses a feature-based registration method, taking the DAPI channel image of each round as the benchmark to calculate the transformation relationship between rounds, and then migrates it to the remaining antibody channels of the same round in the form of a homography matrix 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 distribution characteristics of different antibodies, the overlapping area with specific cell nuclei, the surrounding relationship, and the repulsive relationship between different antibodies. The processing is carried out in a single round and single channel unit, and the cell identity is judged by the relative relationship between the antibody and the cell nucleus in the image. The likelihood probability is given by the overlapping area of ​​effective signals in a given area (intranuclear or perinuclear) (screened by connectivity and grayscale, etc.) and the pixel intensity of the overlapping area, and the likelihood probability under different antibody conditions is compared to give the final cell identity.

[0023] Preferably, the sample in S1 is prepared as one of paraffin sections, frozen sections, and adherent cells.

[0024] More preferably, the specific method of preparing the sample is:

[0025] (1) Tissue cryopreservation

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

[0027] The tissue was rinsed twice with phosphate buffer and dehydrated with 30% sucrose;

[0028] When the tissue sinks to the bottom of the sucrose solution, transfer the tissue into OCT and snap-freeze on dry ice;

[0029] After quick freezing, the tissue was stored in a -80°C refrigerator;

[0030] (2) Slide coating

[0031] The slides were immersed in 1M HCL solution, shaken at 40 rpm for 1 h, and rinsed with water twice, then immersed in 1M NaOH, shaken at 40 rpm for 1 h, and rinsed with water twice;

[0032] The slides were immersed in a 1% bound silane solution at pH 3.5, shaken at 40 rpm for 1 h, and rinsed with ethanol three times;

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

[0034] (3) Slicing

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

[0036] Tissue sections with a thickness of 18 μm were cut;

[0037] Use a brush to carefully spread out the tissue sections;

[0038] Bring the coated slide close to the section so that it sticks to the slide.

[0039] Preferably, the specific steps of S2 are:

[0040] The slide with the tissue section was placed in a 60°C oven for 1 hour; then the slide was fixed in 2% PFA for 20 minutes and washed three times with phosphate buffer, each time for 10 minutes;

[0041] Then, an appropriate amount of blocking solution was added to the tissue sections for 20 minutes; primary antibody was added to the tissue sections, and the sections were covered with sealing film and shaken at 4°C overnight; the blocking solution was a phosphate buffer containing 5% donkey serum and 0.3% Triton-X100;

[0042] Then, the tissue sections were immersed in phosphate buffer for 3 times, each time for 10 min, and then the corresponding secondary antibody was added to the tissue sections and shaken at room temperature for 2 h.

[0043] Then, the tissue sections were immersed in phosphate buffer for 2 times, each time for 10 minutes, and then 1 μg / ml DAPI was added to the tissue sections. After treatment for 10 minutes, the sections were immersed in phosphate buffer for another 10 minutes. The sections were sealed with 65% glycerol solution.

[0044] Finally, the fluorescent staining images were obtained.

[0045] Preferably, in S3:

[0046] The eluent is Aibixin abs994;

[0047] The eluent heating temperature is 25-37°C;

[0048] The shaking time is 5 to 10 minutes;

[0049] The sealing method adopts a plastic bag;

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

[0051] Preferably, the fluorescent staining image is obtained in S2 or S4 by taking pictures using a laser confocal microscope or a common fluorescence microscope.

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

[0053] Grayscale histogram equalization, image morphological operations and median filtering denoising methods are combined to operate pixels and patterns to enhance the local features of antibody images.

[0054] Preferably, the DAPI image recognition is implemented using a pre-trained model of cellpose2.0.

[0055] Contains at least the following beneficial technical effects:

[0056] The present invention can realize in-situ detection of multiple proteins by simply increasing the number of imaging rounds, greatly simplifying the complex reagent configuration, and reducing costs while ensuring the accuracy of experimental results. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 These are three consecutive rounds of immunofluorescence imaging of brain tissue sections from 5xFAD mice.

[0058] Figure 2 This is an overlay of three consecutive rounds of immunofluorescence imaging of brain tissue sections of 5xFAD mice. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0060] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0061] It is obvious to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the main idea or essential features of the present invention. Therefore, from all points of view, the embodiments should be regarded as exemplary and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims are included in the present invention, and any reference numerals in the claims should not be regarded as limiting the claims involved.

[0062] In addition, it should be understood that although this specification is described according to the implementation modes, not every implementation mode includes only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation modes that can be understood by those skilled in the art. These other implementation modes are also covered within the protection scope of the present invention.

[0063] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the protection scope of the present invention / invention.

[0064] Example

[0065] S1. Preparation of sample tissue sections: 12-month-old 5xFAD mice were perfused with PBS and then brain tissue was removed, fixed with 4% PFA overnight, dehydrated with 30% sucrose, embedded in OCT and frozen on dry ice. The sections were sliced ​​in a cryostat and mounted on PDL-coated slides.

[0066] S2. Fluorescence staining: The slides with tissue sections were dried in a 60°C oven for 1 hour, fixed in 2% PFA for 20 minutes, washed 3 times with phosphate buffer, each time for 10 minutes. Then 5% donkey serum was used for blocking for 20 minutes, and then CD68, Aβ and Olig2 antibodies were incubated. After applying the corresponding secondary antibodies, PBS was washed 3 times, each time for 10 minutes, and finally stained with DAPI, washed once with PBS, and photographed. The first round of antibodies was removed using antibody elution solution (Aibixin abs994), and the second round (GFAP, Iba1, AldC) and third round (Sox1, Sox10, Sox9) antibodies were incubated in the same way. After each round, the antibodies were eluted with antibody elution solution (Aibixin abs994), and photographed respectively.

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

[0068] Figure 1 This is a three-round continuous in situ protein staining of 5xFAD mouse brain slices, and the image was taken in the mouse hippocampus.

[0069] Figure 2 This is an overlay of the three rounds of protein staining signals obtained.

Claims

1. A method for tissue in situ protein immunofluorescence cycle staining and data analysis, characterized in that: The following steps are involved: S1. Prepare sample tissue sections; S2. Fluorescence staining; S3. Elution: Heat the antibody eluate and drip it onto the fluorescently stained tissue sections and shake them at room temperature; then remove the antibody eluate, drip it again, seal the slide, and treat it in a water bath; then remove the antibody eluate and wash it with phosphate buffer; S4. Cycle dyeing: After the elution is completed, steps S2 to S3 are repeated according to the detection target protein to perform cyclic staining; after each staining, a fluorescent staining image is obtained; S5. Cell nucleus image recognition: After each round of staining, after the last staining and washing is completed, the tissue sections are stained with DAPI to stain the cell nuclei. After the 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 the cell mask and number, and use the cell nucleus area for quality control screening to eliminate the cell nuclei with incorrect boundary division; S6. Differential enhancement of antibody images: The images of different antibodies obtained by cyclic staining were denoised to facilitate the subsequent division of antibody-positive areas; S7. Multiple rounds of image registration: Multi-round image registration uses a feature-based registration method, taking the DAPI channel image of each round as the benchmark to calculate the transformation relationship between rounds, and then migrates it to the remaining antibody channels of the same round in the form of a homography matrix to achieve spatial registration of antibody signals; S8. Antibody-positive cell screening and connectivity segmentation based on overlapping area: Through the distribution characteristics of different antibodies, different cell identities are divided based on the overlapping area with specific cell nuclei, the surrounding relationship, and the repulsion relationship between different antibodies.

2. The method according to claim 1, characterized in that The sample in S1 is prepared as one of paraffin sections, frozen sections, and adherent cells.

3. The method according to claim 1, characterized in that The specific steps of S2 are: The slide with the tissue section was placed in a 60°C oven for 1 hour; then the slide was fixed in 2% PFA for 20 minutes and washed three times with phosphate buffer, each time for 10 minutes; Then, an appropriate amount of blocking solution was added to the tissue sections for 20 minutes; primary antibody was added to the tissue sections, and the sections were covered with sealing film and shaken at 4°C overnight; the blocking solution was a phosphate buffer containing 5% donkey serum and 0.3% Triton-X100; Then, the tissue sections were immersed in phosphate buffer for 3 times, each time for 10 min, and then the corresponding secondary antibody was added to the tissue sections and shaken at room temperature for 2 h. Then, the tissue sections were immersed in phosphate buffer for 2 times, each time for 10 minutes, and then 1 μg / ml DAPI was added to the tissue sections. After treatment for 10 minutes, the sections were immersed in phosphate buffer for another 10 minutes. The sections were sealed with 65% glycerol solution. Finally, the fluorescent staining images were obtained.

4. The method according to claim 1, characterized in that: In the S3: The eluent is Aibixin abs994; The eluent heating temperature is 25-37°C; The shaking time is 5 to 10 minutes; The sealing method adopts a plastic bag; The water bath treatment temperature is 37-50° C. and the treatment time is 30-60 min.

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

6. The method according to claim 1, characterized in that The noise reduction method in S6 is: Grayscale histogram equalization, image morphological operations and median filtering denoising methods are combined to operate pixels and patterns to enhance the local features of antibody images.

Images