Ethanol, never methanol for CD marker fixation RE: [Histonet] immunostaining post-FACS

[Gayle Callis]

Dear Mark and Melissa,'

A correction to Marks reply.  I do NOT use methanol in my acetone/alcohol 
fixative.  The fixative is made up with absolute ethanol 75% acetone/25% 
absolute ETHANOL.  Methanol ruins my CD markers (in the literature and also 
in Histonet archives) .  However, this acetone alcohol fixative is not good 
with human CD4 and CD8 markers since they do not like ethanol either.  And 
there are antibodies that will work best with formalin or paraformaldehyde 
fixation.

Could it be the antigens you are trying to stain do not like the fixative 
you are using?  I suggest you do a fixation panel on known positive cells 
to optimize the fixation.  That positive control is important to know IF 
your antibodies, and method is working.  A known positive tissue control 
may help too, then you know your antibodies are working on mouse tissues, 
cells.  Frozen sections and different fixatives should provide some 
answers.  Some antibodies work better on Western blots, but when you try to 
use them on tissue sections (or at the same concentration as the blot) you 
get nothing.   Also, we try to purchase antibodies known to work on 
cells/tissue sections, and avoid just the Western Blot application.

Double check your BSA, and make sure it is protease/immunoglobulin free, 
Jackson has this - there is an interesting publication on albumin causing 
background staining in AIMM journal, titled Albumin in 
Immuhohistochemistry:  Foe or Friend?  14(4l ), December 2006 Mittelbronn M 
et al.  I do have the publication in pdf form if you would like to read it.

I am curious and as Mark pointed out - to sort the cells, are you staining 
those with an antibody, sort, collect, then stain with another 
antibody?   I was a bit lost on this.

Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610





At 06:08 PM 8/8/2007, you wrote:
>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
>know.
>
>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
>
>mtarango <@t> nvcancer.org
>
>Direct Line (702) 822-5112
>
>Fax (702) 939-7663
>
>
>
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melissa
>Mazan
>Sent: Wednesday, August 08, 2007 4:16 PM
>To: histonet <@t> lists.utsouthwestern.edu
>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
>Tel:508-839-5395
>Fax:508-839-7922
>email: melissa.mazan <@t> tufts.edu
>
>
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>"EMF <nvcancer.org>" made the following annotations.
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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