1. In the culture plate, soak the slides that have been climbed with PBS three times for 3 minutes each time.
2. Fix the slides with 4% paraformaldehyde for 15 minutes, and soak the slides in PBS for 3 times, 3 minutes each time.
3. Permeabilize with 0.5% Triton X-100 (prepared in PBS) for 20 minutes at room temperature (for antigens expressed on the cell membrane, this step is omitted).
4. Serum blocking: immerse the slides in PBS for 3 times for 3 min each time, dry the PBS with absorbent paper, add normal goat serum (provided that the secondary antibody source is goat) on the slides, and block at room temperature for 30 min.
5. Add primary antibody: Absorb the blocking solution with absorbent paper, do not wash, add a sufficient amount of diluted primary antibody to each slide and put it in a wet box, incubate at 4°C overnight.
6. Add fluorescent secondary antibody: immerse the slides in PBST for 3 times, 3 minutes each time, absorb the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody dropwise, incubate at 20-37°C for 1h in a wet box, soak in PBST Slice 3 times, 3 min each time.
Note: From the addition of fluorescent secondary antibody, all subsequent steps should be performed in a dark place as much as possible.
7. Serum blocking: blot the liquid with absorbent paper, drop normal goat serum on the glass slide (provided that the secondary antibody source is goat), and block at room temperature for 30 minutes.
8. Add primary antibody: Absorb the blocking solution with absorbent paper, do not wash, then add the diluted primary antibody dropwise, and incubate overnight in a 4°C humid box in the dark after adding the primary antibody. (Note: the species of the second primary antibody is different from the species of the first primary antibody, such as mouse and rabbit)
9. Add fluorescent secondary antibody: immerse the slides in PBST for 3 times, 3 minutes each time, absorb the excess liquid on the slides with absorbent paper, add the diluted fluorescent secondary antibody dropwise, incubate at 20-37°C for 1h in a wet box, soak in PBST Slice 3 times, 3 min each time. (Note: If the red fluorescence is selected for the detection of the first antibody, the second one must select other colors of fluorescence)
10. Counterstaining nuclei: DAPI was added dropwise and incubated in the dark for 5 minutes, the specimens were stained, and the excess DAPI was washed off with PBST 5min×4 times.
11. Dry the liquid on the slide with absorbent paper, seal the slide with a mounting liquid containing anti-fluorescence quencher, and observe and collect images under a fluorescence microscope.
