[Histonet] F4-80 immunofluorescence of cells for confocal

From:"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
Any suggestions?

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