Stupid Question(s)?

Calman Prussin (Calman_Prussin@d10.niaid.pc.niaid.nih.gov)
Tue, 22 Aug 95 15:01:48 EST

You have not mentioned what you are doing with your fixed cells. The
responses so far have been oriented towards fixation of cells for later
analysis. However, you mention staining for intracellular antigens. When
staining for intracellulare antigens, I routinely stain for surface markers
AFTER my 4% paraformadehyde fixation. Obviously, some epitopes will be
denatured by the fixation, but these are approximately 10% of the mAbs I
have used after trying about 30 different mAbs to various human CD markers.
I should add that most other investigators doing intracellular staining
surface stain BEFORE fixation.

------------------------ Forwarded Message -----------------------
Date: 8-15-1995 4:56pm
From: {owner-cyto-sendout@flowcyt.cyto.purdue.edu}:unix:niaid
To: calman prussin:10:niaid,
barbara butcher:4:niaid
Subj: Stupid Question(s)?
Also-to: cytometry mailing list <cytometry@flowcyt.cyto.purdue.edu>

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It is said that there are no stupid questions but.....

When labeling leukocyte cell surface antigens with the appropriate
conjugated antibody, is it best to fix (with paraformaldehyde) the cell
suspension:
a) before antibody labeling?
b) after antibody labeling?
c) what concentration of paraformaldehyde (1% final concentration?)
and is it dependent upon the
d) cell type?
e) antigen to which the antibody is specific for?
f) monoclonal vs. polyclonal antibody?

If that wasn't enough for you, add in the variable of labeling intracellular
antigens into the question of timing of fixation.

If there is a review of this in the literature, could you point me to it
please.
Thanks for your time and forthcoming answers.

Dan J. Smith
San Diego CA

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