[Histonet] F4-80 immunofluorescence of cells for confocal

[Martin S.]

Hi All,
 
Has anyone used F4-80 to label cultured macrophages. The staining should
be on the cell membrane but I only get internal staining!? My protocol
was as follows - I was trying to see how different staining and
permeabilisation protocls affected membrane staining;
 
1) Label cells in culture with biotinylated F4-80 (Serotec; 1h, 37oC),
wash PBS, fix 4% PFA in PBS (20min, room temp), permeabilise or not with
0.2% Triton, 2% BSA in PBS or 0.05% saponin, 2% BSA in PBS (20min room
temp), incubate with streptavidin Alexa488
 
2) Fix cells as above, block with 2% BSA, label with F4-80, permeabilise
as above, label with streptavidin Alexa488
 
3) Fix cells as above, permeabilise (as above) or not, label with F4-80,
wash, label with streptavidin Alexa488
 
There was no staining on any of the cells without permeabilisation. All
cells with permeabilisation were stained but all staining was
intracellular.
 
 
Any suggestions?