[Histonet] fixation post-fixation, difference of order used

kemlo kemlo <@t> f2s.com
Sat Dec 6 05:02:40 CST 2008

Interesting question.....

Post fixation is sometimes carried out to enhance staining. I the old days
we fixed in formalin, then in mercury. If you fixed in mercury from the
start then the tissue went hard whilst you could leave tissue in formalin
for ages; you can therefore control the exposure time in mercury more

Logically the order of fixation and the types of fixative used must make a
difference; if you initially fixed in mercury too long then in formalin
you'd end up with hard tissue.

Fixatives are there to make the tissue ready for processing, so fixatives
may also make the tissue ready for being fixed.

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
ejschmid <@t> ucalgary.ca
Sent: 05 December 2008 17:30
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] fixation post-fixation, difference of order used


I was wondering about the effects of using different fixatives in
 different orders. For example, some of our embryonic material (chick and
 mouse) is fixed initially with 4% PFA and subsequently is exposed to
 glutaraldehyde. Chick embryos subject to this are successfully labeled
 using IHC for anti-HH3.

However, our mouse tissues are fixed differently, being first exposed to
 4% PFA: 1% Glut solution (Which i think is called a modified Karnovsky's
 fix). These embryos perform poorly for IHC with anti-HH3.

Might it be the glutaraldehyde that is present in our primary fixative
 used to fix moue embryos? Might it be that the IHC works for chick because
 the glutaraldehyde is only used as a secondary fix after primary fixation
 in 4% PFA? Does the order of exposure matter?


eric schmidt

University of Calgary
Medical Sciences

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