贴壁细胞的免疫荧光染色方法
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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.