Fixing cells before staining of surface markers - (Feb/07/2011 )

Hi,

I need to fix cells immediately after stiumaltion to preserve a phosphorylation status.
Then, I would like to stain for surface markers.
Then, permeabilize the cells, maintaining fluorescence on the surface.
At last, stain for intracellular marker (a phospho-protein).

In my experience, fixing the cells first (4% PFA) reduces the ability of the fluorescent antibody to detect correctly the surface marker (the fraction of positive cells is reduced by more than a half).
Nevertheless, I need to first fix the cells, is there an alternative for 4% PFA, in which the esurface staining could be working well?
Please notice taht later on, I would also like to permeabilize cells for inracellualr staining.

I'm familiar with the CytoFix/CytoPerm kit of BD that enables co-staining of surface and intracellualr marker, however, in this method, non-fixed cells are first stained for surface markers and then they are fixed+permeabilized for intracellualr markers, maintaining surface fluorscence.

The disadvantage is obviously that cells are not fixed immediately.

I know this is complicated, but please advice.

4% of PFA is too much. Try 1-2% of PFA with the fixation at melting ice, for 15-20 minutes.
The surface staining after the fixation is a big challenge and for many surface markers it is impossible to have a good staining.