To stain cultured cells we routinely fix with 4% paraformaldehyde for
15-20 mins. But if the antigen is sensitive to aldehydes, then we fix
with ice-cold 100% methanol for 5-10 mins. Your acetone technique for 30
mins is extremely harsh for cultured cells. If you want to continue with
it, I would fix with the acetone for only a few minutes and try that. 

As for the other person who asked about staining cultured cells, we do
exactly the same ICC as on sections, although we usually are able to
dilute the primary antibodies more than with frozen tissue sections
(using paraformaldehyde perfused tissues).

Sarah Pixley
Ohio


Message: 4
Date: Mon, 31 Jul 2006 13:41:54 -0400
From: Christopher C Overend 
Subject: [Histonet] IFA staining problems
To: histonet@lists.utsouthwestern.edu
Message-ID: <1fbaf901fb5b0e.1fb5b0e1fbaf90@huskymail.uconn.edu>
Content-Type: text/plain; charset=us-ascii

I am new to the technique of immunofluorescence, and have been doing
some work with it lately on cultured cells. Specifically, I have been
staining intracellular virus with very good results. However, when i try
to detect cellular proteins, I do not get any staining. The proceedure i
have been using consists of "fixation" with 90% acetone for 30 min, at
4degrees C. the rest of the process had been much like an ELISA, all
incubations have been for 30 min at 4degrees. The buffer i have been
using is PBS, 1%BSA, 0.1%NaN3. Someone mentioned the problem could be
from the acetone and suggested trying a glyoxal fixative. Can anyone
offer some insights, or if there is a different protocol i might have to
follow using a glyoxal fixative with tissue culture?
Thank you!
Chris

Christopher Overend
Ph.D Student
University of Connecticut
Department of Pathobiology and Veterinary Sciences Storrs, CT
Christopher.overend@uconn.edu

 

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