Soluble GFP needs to be fixed
in place before permeabilization.
-
Fix cells in 4
% paraformaldehyde/PBS at 37oC for 5 min. (I don't know
if this is the best fixation. I have never tried different conditions because
I got a reasonable result with this.)
-
Wash once or twice with PBS/1% FCS/0.1 % sodium azide (FACS staining buffer).
-
Suspend the cells in PBS/1% FCS/0.1 % sodium azide/0.1 % saponin (Permeabilization
buffer)
-
Incubate 5 min on ice, and spin.
-
Remove supernatant and stain with antibody diluted in permeabilization
buffer.
-
Wash with permiabilization buffer.
-
Resuspend in FACS staining buffer and analyze.
It is always better to use fresh PFA solution.