[Histonet] IHC staining weaker in...

[Lee & Peggy Wenk]

Another variation on the theme -

Underfixed tissue, so only the outside is fixed with the fixative, and the
center is unfixed.

Then the center is "fixed" with the alcohol on the tissue processor. 

So there are two different fixations going on. Most stains are designed for
the charges on the tissue caused by formalin fixation. If tissue is
primarily alcohol fixed, then the charges on the tissue are not the same as
formalin fixed tissue, so the staining is different.

Either make the tissue thinner (2-3 mm) and/or increase the time in the
fixative. Either way, get the fixative into the center of the tissue AND
allow it time to cross-link, BEFORE the tissue goes into the alcohol step.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Monfils,
Paul
Sent: Thursday, September 06, 2007 12:34 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] IHC staining weaker in...

What is your standard fixative?  Have you made any changes in routine
fixation recently?  This sounds like a fixation problem.  As a core research
facility, we receive all kinds of tissues from various researchers, often in
a variety of fixatives. I have noticed the artifact you describe in some
tissues fixed in certain slow-penetrating fixatives.  Some aggressive
fixatives give very nice preservation in small tissue samples, but when
larger tissues are immersed in such fixatives, the rapid fixation of the
outer layer of the tissue actually sets up a barrier to penetration of the
fixative into the deeper levels. The result is a poorly fixed interior with
a very well fixed exterior, and the poorly fixed deeper areas often stain
poorly.


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