Wash with PBSM (=PBS + 5mM MgCl2) (3 quick rinses)
Fix cells 10 min RT in 4% Paraformaldehyde in PBSM
Wash with 0.1M Glycine/PBSM 10 min @ RT (quenching)
Wash with PBSM 2 x 10 min @ RT (cells can be left overnight @ 4°C at this point).
Permeabilize cells 5 min with PBS supplemented w/ 0.2% triton x100. (0.2% PBT)
Blocking: incubate for 30' in 0.1% PBT with 3% BSA.
Place coverslip over 50μl of primary Ab diluted in 0.1% PBT with 3% BSA in a humid chamber for 2h at RT.
Wash 3x5' with PBT 0.1%.
Place coverslip over 50μl of second Ab diluted in 0.1% PBT with 3% BSA in a humid chamber for 20-30 min at RT.
Wash 3x5' with PBT 0.1%.
Wash 5min in PBS.
Mount coverslip onto a slide using mounting medium (prolong gold+DAPI).
Note: Always have negative control of the secondary antibody (without the primary antibody)