Re: Fixation prior to staining
Becky Bonner (Becky.Bonner@CCLINK.NET.uokhsc.edu)
Thu, 30 Jun 94 15:11:13 CST
We have had a lot of luck with most biomarkers being preserved by
using 0.5% paraform. The past few years we have been using
PolySciences EM grade formaldehyde and usually can't find any
difference between the two reagents (the Polysciences product is
reallly expired paraformaldehyde since paraform readily and quickly
converts to form in solution). This is a much more convenient method
and not hazzardous like paraform is to make. I wish I could remember
who recommended I try the form ... I didn't have much confidence that
it would work but tried it anyway ... now I can't remember who it was
but would like to thank them if they read this email. It has
revolutionized our clinical sample collections. We were running with
ice and sneakers sometimes in the middle of the night and weekends to
collect patient samples.
Our standard protocol is the following: We usually treat our cells
with 0.5% EM formaldehyde for 15 min. followed by 25% non-fluorescing
ethanol. The addition of the ethanol reduces the nonspecific signal
you are describing in the orange and red range cold ethanol helps
reduce this even further ... (why?????). The formaldehyde crosslinks
the proteins of interest while preserving their antigenicity. The
higher the formaldehyde/paraform the more non-specific signal since
there is more crosslinking and more difficult to get things in and out
of the cell. Concentrations of form > 1% deteriorate CV's of DNA and
significantly impact nonspecific orange/red signal. Some nuclear
antigens may not be accessible after crosslinking but otherwise most
antibodies seem to like this method.
______________________________ Reply Separator _________________________________
Subject: Fixation prior to staining
Author: drwho@pandoras-box.bgsm.wfu.edu (Robert Rainer) at cclink
Date: 6/28/94 12:19 PM
Hello Fellow Flow Heads;
I have been playing around with fixation of human cells prior to
staining with antibodies. I have tried 1% paraformaldehyde 50% and 70%
ETOH and Acetone. Neither of these techniques is very satisfactory. The
paraform worked the best, but I did see a sit in the orange auto
flouresence, but the antigenicity was preservered. Also there appeared to
be a decrease of the green signal. I am sure that people have worked on
this in the past, and I was wondering what other methods people have used,
and what kinda results were obtained.
Has anyone tried any methods of antigen retrival using microwaves
or boiling? Any suggestions would be great, and thanks for your time -- rr
---
Robert Rainer, M.D.
Department of Pathology drwho@pandoras-box.bgsm.wfu.edu
BGSM
Winston-Salem, N.C. 910-716-4312
27157
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