RE: [Histonet] immunostaining post-FACS

From:"Tarango, Mark"

These cells haven't already been stained and sorted or something, have
they?  If so, I would assume that whatever antibodies they used for
flow/FACS will be on the cells.  You said you use cytofix/cytoperm, that
might denature the flow antibodies (if there are any), but you never

Have you tried doing the cytofix/cytoperm on the cells while they are in
suspension, and THEN making the cytospins?  Switching to a different
fixative?  Gayle Callis had a post a while ago about using a fixative of
75% acetone and 25% methanol for murine CD markers.  Maybe it would work
for all your markers.  I've tried modifying this fixative using propanol
instead of ethanol for frozen sections on human tissue.  It worked.

When all else fails, I like to, as closely as possible, treat the
specimen like formalin-fixed paraffin-embedded tissue.  I will fix in
formalin, dehydrate in staining dishes (like processing tissue) ...then
bring it back to water and do antigen retrieval before staining.  You
might lose too many cells trying this on cytospins though, but might as
well throw it out there.

Just some thoughts...

Mark Adam Tarango HT(ASCP)

Histology/Immunohistochemistry Supervisor

Nevada Cancer Institute

One Breakthrough Way

Las Vegas, NV  89135

Direct Line (702) 822-5112

Fax (702) 939-7663


-----Original Message-----
[] On Behalf Of Melissa
Sent: Wednesday, August 08, 2007 4:16 PM
Subject: [Histonet] immunostaining post-FACS

Hi all, Wondering if anyone has done much immunostaining post FACS.  WE 
have had a lot of trouble with this procedure - we collect our cells 
(from murine lung) into BSA on ice,  bring them back to our lab (FACS 
is at our core facility, so it means about an hour and a half before 
the cells get to our lab).  In the lab, we immediately cytospin - the 
morphology is nice on H and E - and then fix in Cytofix Cytoperm for 20 
minutes at 4C.  We either commence immunostaining immediately after, or 
leave in fridge for the next day. We do either immunofluorescence or 
immunoenzyme (usually ABC system) for identifying antigens of interest. 
  We always have a negative control, but don't always have a positive 
control.  We do either double staining for SPC and CC10 or single 
staining for alpha sma, vimentin, e-cad, or pancytokeratin.  The 
problem that we have is that although the negative controls are very 
negative with respect to the cases, we seem to be getting a lot of 
false positives when the antibody is actually applied - for instance, 
my cells will stain 80% positive - strongly positive - using alpha sma, 
but a Western of the same cells will show no alpha-sma at all, whereas 
there is positive staining on whole lung suspensions.  Any ideas? 
Advice for getting good post-FACS immunostaining? Many thanks - Melissa

Melissa R. Mazan, DVM, Diplomate ACVIM
Associate Professor and Director of Equine Sports Medicine
Department of Clinical Sciences
Tufts Cumming School of Veterinary Medicine
200 Westborough Road
North Grafton, MA 01536

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