RESOURCES
A. Required solutions and reagents
1. Phosphate buffer: 0.1M 1X PBS (pH 7.4)
2. Fixing solution: 4% paraformaldehyde solution (no methanol)
3. Antibody dilution: 1% BSA, 0.03% Proclin300 in 0.1 M PBS
4. Antigen repair solution: sodium citrate buffer
5. Closed normal goat serum (stock solution)
6. DAPI dyeing solution
7. Immunofluorescence anti-quenching tablets
B. Steps
1. 60 ° C baked 2 hours
2. Dewaxing. Dewaxed in xylene for 15 minutes, three times
3. Hydration. Pass through absolute ethanol, 95%, 90%, 80%, 70%, each 5 minutes, distilled water (or
Come water) 5 minutes
4. Wash PBS for 5 minutes, three times
5. Antigen retrieval. Boiling in citrate buffer (about 92-95 ° C)
Bath) Repair for 15 minutes, naturally cool to room temperature. Wash in PBS for 5 minutes, three times
6. Normal goat serum is blocked, 37 ° C for 20 minutes
7. Wash PBS for 5 minutes, three times
8. Incubate with primary antibody and act for 1.5 hours at 37 °C (concentration of incubation antibody, time of action through pre-experiment
Decide).
9. Wash PBS for 10 minutes, three times
10. DAPI counterstained nuclei, diluted 1:1000, for 5 minutes at room temperature.
11. Wash in PBS for 5 minutes, three times
12. Anti-quenching fluorescent sealant seals, observed under the microscope.
