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We are using Ortho's PermeaFixt (#775999) for our surface labelling and in situ
hybridization of human PBMC. We surface label (tagged-primary antibodies) prior
to fixation, then hybridize with either biotinylated oligos or riboprobes, and
then analyze by flow. We have not done any sorting as yet. We find that this
product preserves the integrity of the cells and does not impact too heavily on
the surface staining. Its expensive, but it seems to do the job. We monitor
permeability with a biotinylated polydT control.
good luck!
Marty Giedlin
Chiron Corp.
Marty_Giedlin@cc.chiron.com
510-420-6386
_______________________________________________________________________________
Subject: Fixation of human lymphocytes
Author: Marty Giedlin
Date: 1/11/95 9:23
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Date: Tue, 10 Jan 1995 11:54:46 -0700 (MST)
From: "In C. Kim" <inkim@unm.edu>
Subject: Fixation ofhuman lymphocytes
To: cytometry@flowcyt.cyto.purdue.edu
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Hi all,
I have a basic question on how to fix cells.
We normally isolate lymphocytes and granulocytes from human blood by using
Histopaque centrifugation, and wash with PBS to remove absorbed
Histopaque. These cells are labelled with primary antibodies, and then
with secondary antibodies before flow sorting. We use these sorted cells
for in situ hybridization.
It would be better if we could fix cells before labelling with antibodies.
Fixation of cells with paraformaldehyde requires a proteolytic digestion
step prior to in situ hybridiztion. Therefore, we are avoiding
formaldehyde fixation.
We tried to fix cells with methanol or ethanol (70% to 100%) before
labelling with antibodies. The problem is that cells undergo aggregations
by alcohols. Could somebody suggest how to avoid the aggregation during
fixation or other alternative, gentler fixation methods?
Thanks.
In C. Kim <inkim@carina.unm.edu>
University of New Mexico School of Medicine
Albuquerque, New Mexico
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