Protocol of Immunofluorescence (IF) on attached cells

Materials:

1. PBS solution

2. 4% Paraformaldehyde (PFA fixative):

Dissolve 4g paraformaldehyde in 100ml PBS solution, stir at 70℃ to dissolve;

3. PBS-T solution: (0.1% Triton X-100 in PBS solution)

4. PBS-B blocking solution: (4% BSA in PBS solution)

5. Primary antibody: Dilute with PBS-B solution, dilution factors should refer to manual, or, test from 1:50~200, should be more concentrate than application in Western blot;

6. Secondary antibody: Dilute with PBS-B solution, dilution factors should refer to manual

Procedure:

1. Cultured cells, let it attach to the coverslips in 6-well plate;

2. Remove medium, rinse with PBS twice;

3. Add 2ml 4% paraformaldehyde, incubate at room temperature for 20 minutes;

4. Rinse with 2ml PBS three times, rinse for 5 minutes every time;

5. Permeabilize cells with 2ml PBS-T solution at 4?C for 10 minutes;

6. Remove PBS-T solution, rinse cells with PBS for 5 minutes at room temperature;

7. Block non-specific interaction with PBS-B solution at 37?C for 30 minutes;

8. Add primary antibody solution, incubate at 4?C overnight;

9. Remove primary antibody solution, wash with PBS for 5 minutes;

10. Add secondary antibody solution, incubate at room temperature for 1 hour;

11. Wash with PBS three times, 5 minutes each;

12. Add anti-fade DAPI solution if needed;

13. Observation.