[Histonet] immunostaining post-FACS

From:Melissa Mazan

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
email: melissa.mazan@tufts.edu

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