IF staining protocol

– 4% PFA: dissolve 0.4g PFA powder in 10ml PBS, heating to help dissolve;
– Permeabilization buffer: 0.2% Triton X-100 in PBS;
– Blocking buffer: 3% BSA, 0.1% Triton X-100 in PBS;
– Washing buffer: 0.1% Triton X-100 in PBS;

1. Place cover slides in 24-well plate, coat with Matrigel;
2. Seed cells onto cover slides in 24-well plate, incubate for at least 24h to let cell attach and grow on the slides;
3. Aspirate medium, wash once with PBS, fix with 4%PFA, RT 10-15min;
4. Wash with PBS, at least two times (the cover slides can be store at 4℃ for up to 1 week);
5. Incubate in permeabilization buffer (0.2% PBST), 10min @RT;
6. Incubate in blocking buffer (3% PBSA), 30min @RT;
7. Incubate with primary antibodies diluted in blocking buffer, 2h @RT or 4℃ O/N;
8. Wash 3x5min with washing buffer;
9. Stain with DAPI, 1min @RT;
10. Mount the cover slides on a glass slide with Vectashield or prolong gold mounting medium, seal with nail polish, air dry for 15-30min to let the nail polish solidify completely.
11. store glass slides at 4℃ for short term and -20℃ for long term.