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. 0.3% cell penetrating agent : 0.3% Triton X-100
5. Normal goat serum (stock solution) for closure
6. DAPI dye solution
B. Steps
1. Discard the cell culture medium and wash 3 times with PBS for 5 min/time.
2. Fixation: 4% paraformaldehyde is fixed at room temperature for 20 min, or fixed at 4 °C overnight.
3. Wash 3 times with PBS for 5 min/time.
4. Permeation: 0.3% Triton X-100 for 20 min at room temperature (if the detected protein is fine)
Membrane protein, this step can be omitted).
5. Wash 3 times with PBS for 5 min/time.
6. Blocking: 1:20 diluted goat serum (secondary source serum) was blocked at room temperature for 20 min.
7. Wash 3 times with PBS for 3 min/time.
8. Primary antibody 1:200 dilution, 1.5 h at 37 °C (concentration of incubation antibody, effect time by pre-experiment set).
9. Wash 3 times with PBS for 10 min/time.
10. DAPI counterstained nuclei, diluted 1:1000, and allowed to stand at room temperature for 5 min.
11. Wash 3 times with PBS for 5 min/time.
12. Remove the cell slides and place them on a glass slide and observe under the microscope.
