RESOURCES
A. Required solutions and reagents
1. Phosphate buffer : 0.1M1XPBS (pH 7.4)
2. Fixing solution: 4% paraformaldehyde solution (no methanol)
3. Antibody dilution : 1% BSA, 0.03% Proclin 300 in 0.1 M PBS
4.0.3% cell penetrating agent : 0.3% TritonX-100
5. Closed normal goat serum (stock solution)
6.DAPI dyeing 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 PBS 3 times, 5 min / time.
4. Permeation: 0.3% TritonX-100 at room temperature for 20 min (if the detected protein is fine Membrane protein, this step can be omitted).
5. Wash PBS 3 times, 5 min / time.
6. Blocking: 1:20 diluted goat serum (secondary source-derived serum) was blocked at room temperature for 20 min.
7. Wash PBS 3 times, 3 min / time.
8. Primary antibody 1:200 dilution, 1.5h at 37 °C (incubation of antibody concentration, action time through pre-experiment set).
9. Wash PBS 3 times, 10 min / time.
10. Secondary antibody 1:300 dilution, 1 h at 37 °C (incubation of antibody concentration, action time by pre-experiment Fixed).
11. Wash PBS 3 times, 10 min / time.
12. DAPI counterstained nuclei, diluted 1:1000, and allowed to stand at room temperature for 5 min.
13. Wash PBS 3 times, 5 min / time.
14. Remove the cell slides and place them on a glass slide and observe under the microscope.
