Re: Ethanol fixation

Adrian Moris (Adrian_Moris@sratech.com)
Wed, 11 Sep 96 10:48:07 EST

I'm fairly new to the flow cytometer, however, in my previous
capaicty, I was involved with staining transformed cells that had been
air dried to 96 well flat bottom plates. This process involved
fixation with a acetone/methanol (50/50, V/V) solution. This opened
up the membranes for intracellular staining.

The method, for the 96 well plate method is as follows:

1. Add 200ul A/M to each well for 2 minutes. Be certain the cells do
not dry out.

2. Remove the A/M and wash with PBS.

3. Repeat the wash twice.

I feel certain this method could be adapted for use with flow
cytometry. Please note that fixation by this method can destroy some
antigens, making binding weak or impossible. However, for most
applications, I found it to work quite nicely.

Good Luck. Let me know what you come up with.

Adrian Moris

______________________________ Reply Separator _________________________________
Subject: Ethanol fixation
Author: "Bruce S. Hass" <BHASS@nctr.fda.gov> at Internet
Date: 9/10/96 10:50 PM

Hi,
I am interested in staining cells with Bodipy by the method of Gocze
and Freeman (Cytometry 17:151-158 1994), which calls for ethanol
fixed cells. Unfortunately the paper does not give details about the
fixation procedure. Is ethanol fixation so simple, obvious, or common?
Any suggestions on how to do it would be appreciated. Thanks.

Bruce Hass
National Center for Toxicological Research
Jefferson AR 72079
(501) 543 7365
(501) 543 7745 (fax)
bhass@nctr.fda.gov

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