Guide to immunofluorescence experiments
Guide to immunofluorescence experiments
Fixation and permeabilization
1. Wash the coverslip with 1 × PBS 3 times for 3 min each in a petri dish with the cells that have climbed.
2. Fix the climbing slice with 4% paraformaldehyde for 15 min, wash the slide with 1 × PBS 3 times for 3 min each.
3. 0.5% Triton X-100 (prepared in 1× PBS) permeabilize the cells at room temperature for 15 min (omit this step for antigens expressed on the cell membrane), and rinse the slides three times with 1× PBS for 3 min each.
Block with
4. absorbent paper to dry 1 × PBS, add 5% normal serum (same or similar species as the secondary antibody) to the slides by dropwise addition and seal at room temperature for 1 h.
Antibody incubation
5. Absorbent paper to remove the blocking solution, no washing, add enough diluted primary antibody to each slide and place in a wet box, incubate overnight at 4 °C.
6. Add fluorescent secondary antibody: Soak the slides in PBST three times for 3 minutes each time, blot the slides dry with blotting paper, add the diluted fluorescent secondary antibody, incubate in a wet box at 37°C for 1 hour, and then soak the slides in PBST three times for 3 minutes each time.
Note: From the addition of fluorescent secondary antibody onwards, all subsequent steps should be carried out in a dark place as much as possible.
Fix and photograph
7. Re-stain the nucleus: add DAPI dropwise and incubate in the dark for 5 min to stain the nucleus of the specimen. Wash off the excess DAPI with PBST for 5 min × 4 times.
8. Blot the liquid on the slide with absorbent paper, seal the slide with a cover slip solution containing an anti-fluorescence quencher, and observe and collect images under a fluorescence microscope.
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