Immunofluorescent Staining

1. Fix cells in 4& PFA (cold) for 30min, RT
2. Wash cells with PBS Buffer for 5min, 3x
3. Incubate cells in 0.3% Triton-X 100 for 5 min, RT
4. Wash cells with PBS Buffer for 5min, 3x
5. Incubate cells in 5% FBS + 1%BSA in PBS for 30min, RT
6. Incubate cells with the first primary Ab for 1h, RT
7. Wash cells with PBS Buffer for 5min, 3x
8. Incubate cells with first secondary Ab for 30min in the dark, RT
9. All the following steps should be incubated in the dark.
10. Wash cells with PBS Buffer for 5min, 3x
11. Incuabate cells in 5%FBS+ 1%BSA in PBS for 30min, RT
12. Incuabate cells with 2nd primary Ab for 1h, RT
13. Wash cells with PBS buffer for 5 min, 3x
14. Incubate cells with the 2nd secondary Ab for 25min, RT
15. Add DAPI (final conc 300nM) to the 2nd secondary Ab incubation for 5min, RT
16. Wash cells with PBS Buffer for 5min, 3x
17. Mount cells with fluorescence mounting media. Store in the dark at 4C.

Notes:
- primary Ab diluted 1:100 in 5%FBS+ 1%BSA in PBS
- secondary Ab diluted 1:500 in 5%FBS+ 1%BSA in PBS
- All volumes 200ul