Soluble GFP needs to be fixed in place before permeabilization.
  1. 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.)
  2. Wash once or twice with PBS/1% FCS/0.1 % sodium azide (FACS staining buffer).
  3. Suspend the cells in PBS/1% FCS/0.1 % sodium azide/0.1 % saponin (Permeabilization buffer)
  4. Incubate 5 min on ice, and spin.
  5. Remove supernatant and stain with antibody diluted in permeabilization buffer.
  6. Wash with permiabilization buffer.
  7. Resuspend in FACS staining buffer and analyze.
It is always better to use fresh PFA solution.