Re: [Histonet] F4-80 immunofluorescence of cells for confocal

From:Gayle Callis

Since F4/80 is a cell surface marker, you don't need to permeabilize cells 
to achieve positive staining.  I think your fixation with 4% 
paraformaldehyde may be too long, try 2 minutes or do a time panel starting 
at 2 minutes and work up.  You can also do this with the fixative 
concentration especially for cultured cells. We never use aldehyde fixation 
for this marker, but it is in Histonet archives that it works on formalin 
fixed paraffin sections AFTER a retrieval or digestion.  You may have 
crosslinked your antigen too strongly so it requires a longer staining time 
than 1 hr.  We use RT for our staining protocols, and it only takes 30 
minutes for this primary antibody.

We would fix the cells with 75% acetone/25% absolute ethanol at RT for 5 
minutes, go directly to a buffer after fixation (no more air 
drying).    One can air dry the cultured cells before fixation, but rinse 
away the culture media with red indicator (it will fluoresce) and protein 
carrier before air drying or fixation.  We have not found that BSA is not a 
good block with this antibody, you are better off to use 5% goat or some 
other normal serum (horse, swine, donkey)  and add 1.25% mouse serum, use 
this Normal serum block is also the diluent for your biotinylated primary 
also (rat antimouse monoclonal).  Our normal serum block is 30 minutes 
before applying the primary.

The Strepavidin 488 should be in a serum free diluent.  We prefer a buffer 
with a very low deteregent concentration for immunofluorescence staining. 
0.025% Tween 20 to all diluents, buffers, leaving out BSA or a normal serum 
entirely in an immunofluorescent staining protocol with murine CD markers 
has proven very adequate, clean. Buffer can be either TBS or Dulbeccos PBS 
with 0.025% Tween 20, and we use this to dilute the Strepavidin 488, or 
simply use pure buffer to dilute the SA-488.  Molecular Probes advises no 
serum in diluent with any Strepavidin Alexa Fluors.

Question:  are these cells supposed to be positive for F4/80, as there are 
other macrophage CD markers?  You may need to use another antibody?

Saponin is a detergent generally used for intercalating the cholesterol 
component of cell walls and used for intracellular immunoCYTOchemical 
staining for cytokines attached to the Golgi in single cells.

a At 08:45 AM 12/6/2006, you wrote:
>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?
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)

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