[Histonet] F4-80 immunofluorescence of cells for confocal

[Martin S.]

Thanks, that's v helpful!

The cells were positive for F4-80 by flow cytometry. Basically I want to
look at F4-80 labelling along with an intracellular protein so I need to
be able to permeabilise the cells without affecting the membrane
staining. I will try changing the fixation protocol. Interestingly the
only time I have seen good membrane staining with the F4-80 was when I
labelled unfixed cells with F4-80 followed by SA-488 and THEN fixed the
cells with 4% PFA.

I have been using 4% PFA as most of the articles I have looked at use it
and I used to use it when looking at nuclear proteins but I havent done
much work with membrane proteins so I guess I just have to experiment.

Thanks
Sonya

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 06 December 2006 16:31
To: Martin S.; Histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] F4-80 immunofluorescence of cells for confocal

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 
>intracellular.
>
>
>Any suggestions?
>
>
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)