Cells
Immunofluorescence protocol

Do not let cells dry up, keep in liquid at all times until end.

1. Wash off Media with 3 washes of 1xPBS
2. Fix in 2% paraformaldehyde for 15 minutes, made in PBS.
3. Permeabilize with 0.1% Triton X made in PBS solution for 15 min.
4. Rehydrate with 3 washes of PBS.
5. 5 washes of .5% BSA.
6. BLOCK with 2% BSA for 45 minutes.
7. 5 washes of .5% BSA.
8. Primary antibody in .5% BSA for 60 minutes. (Vortex and Spin Down**)
9. 5 washes of .5% BSA.
10. Secondary antibody in .5% BSA for 60 minutes. (Vortex and Spin Down**)
11. 5 washes of .5% BSA.
12. 5 washes of PBS.
13. Hoescht stain for 30 seconds.
14. 2 wash with PBS.
15. Cover slip with gelvatol and refrigerate.
16. Slides or coverslips must dry overnight at 4 C in the dark.

2% BSA = 2g per 100mL 1xPBS

0.5% BSA = .5g per 100mL 1xPBS

** Spin Down in Microcentrifuge at Maximum Speed for 5 Minutes