[Histonet] Cell permeabilization and cell surface staining

[Martin S.]

Hi all,
 
I have been staining BMDC's to look at proteins which are present on the
cell surface and in the cytoplasm. I fix the cells with 4%
paraformaldehyde in PBS, 20min on ice and permeabilize with 0.2% Triton
X100 in PBS for 20min at room temp. My two problems are:
 
1) When I dont permeabilize the cells I can still detect internal
antigen with my primary antibody (cells are fixed 4% PFA, washed PBS and
blocked with 2% BSA before incubation with antibody)
 
2) When I permeabilize the cells I get a decrease in the cell surface
staining
 
Any suggestions greatly appreciated.
 
BW
Sonya