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 0.1 M PBS
4. Antigen Repair Solution : Sodium Citrate Buffer
5. Normal goat serum (stock solution) for closure
6. DAPI dye solution
7. Immunofluorescent anti-quenching sealant
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. Citrate buffer (about 92-95 ° C) boiled (boiling water)
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. Incubate with primary antibody at 4 ° C overnight.
8. Rewarming for 20 minutes, washing with PBS for 5 minutes, three times
9. Incubation by secondary antibody, 1.5 hours at 37 °C (concentration of incubation antibody, time of action through pre-experiment
Decide).
10. Wash PBS for 10 minutes, three times
11. DAPI counterstained nuclei, diluted 1:1000, for 5 minutes at room temperature.
12. Wash PBS for 5 minutes, three times
13. Anti-quenching fluorescent sealant seals, observed under the microscope.