[Histonet] RE: Acetone fixation problems with OCT Tissues

[Connolly, Brett M]

Patrick,

We do a lot of frozen section IHC work. Years ago Gayle Callis turned me on to fixing in cold acetone:ethanol (3:1) . We keep it at -20C and I fix for 10  min. on the bench then wash in PBS and proceed with the IHC. We do dry slides for at least 30 min before fixing.  This has worked well in our hands for many different antibodies.

Brett

Brett M. Connolly, Ph.D.
Principle Scientist, Imaging Dept.
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
brett_connolly <@t> merck.com
T- 215-652-2501
F- 215-993-6803



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Lewis, Patrick
Sent: Tuesday, April 28, 2015 5:56 PM
To: (Histonet <@t> lists.utsouthwestern.edu)
Subject: [Histonet] Acetone fixation problems with OCT Tissues


Hi Everyone,

I am still having issues with my IHCs with Acetone fixation.

If I fix in 100% Acetone, I get IHC staining, but my tissues are 50-90% destroyed.

If I fix in 4% paraformaldehyde, or 10% NBF or (95%  Etoh and/or Methanol with Acetone) I lose the epitopes I either get no staining or very  weak staining, but the tissue morphology look fine.

I just tried an acetone gradient where I cut the tissues at 5 uM and dried them overnight, then fixed for 10 minutes in 100% acetone, then fixed in 95% acetone for 1 minute, then fixed in 70% acetone for 30 seconds, then quick rinsed in H20, then washed as normal in DPBS pH 7.4.

I did 4 slides, 2 slides with one company's Charged slides ,and 2 slides with another company's charged slides.

One company's slides look completely destroyed, the others may turn out, it was hard to tell how much damage there was.  I'll know tomorrow when I finish staining and Hemotoxylin them.



Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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