[Histonet] RE: perfusion stiffness

[James Watson]

Watch the liver, it will change color as the blood is washed out.  Are you perfusing first with PBS to rinse the blood out, if you start with formalin small blood vessels and capillaries can become blocked by the blood that has coagulated when exposed to the formalin.  We actually flush with 10% sucrose first since brains flushed with PBS can have up to 20% shrinkage.

I have not heard of a good theory behind body stiffness after perfusion yet, pretty sure it is not the effect of actual cross linking since that takes time.  Is someone knows the cause please let me know.

James Watson HT  ASCP
GNF  Genomics Institute of the Novartis Research Foundation
Scientific Technical Leader II, Histology
Tel    858-332-4647
Fax   858-812-1915
jwatson <@t> gnf.org

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of David Wright
Sent: Wednesday, April 15, 2015 2:06 PM
To: histonet <@t> lists.utsouthwestern.edu
Cc: Yves Heremans
Subject: [Histonet] RE: perfusion stiffness

Hi Yves & Histonet

It is certainly a good sign if limbs etc are stiff after perfusion, but maybe not a guarantee that the target organ is perfect given the short perfusions you describe. Definitely, if I don't see stiffness I worry, check for a torn aortic arch (you are doing it transcardially, I presume), adjust the needle placement and run more fixative until everything is stiff.

Do you harvest organs from all over the body? You can improve the efficiency of perfusion by limiting it to the regions of interest. For example, I perfuse rats for brain extraction and clamp off the descending vessels (clamped to the spine) at the level of the diaphragm. The lower half of the body then doesn't get rigid, but the upper half does so more fully/faster. (In rats, I perfuse for much longer than you describe and post-fix too.) Note there's two kinds of stiffness - an immediate, zombie-like outstretching of the forelimbs (& tail wiggling if you do the whole body) which is immediately satisfying as a sign of good needle placement but only happens with a very fresh cadaver, and a more generalized, slower rigidity. For my brains, I check for neck muscle rigidity as well as the forelimb zombie effect.

best wishes - David
==
David A. Wright, Ph.D.
University of Chicago
Section of Neurosurgery, MC3026
________________________________________
 Histonet Digest, Vol 137, Issue 17 Message: 8
Date: Tue, 14 Apr 2015 09:26:00 +0200
From: Yves Heremans <Yves.Heremans <@t> vub.ac.be>
Subject: [Histonet] transcardial fixation
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <51D52790-789D-4189-92B3-630B6AAD93AE <@t> vub.ac.be>
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Dear Histonetters,

We routinely perfuse mice with fixative before taking out organs. Perfusion with fixative (4% PFA or 10% NBF) is done for only a few minutes (max. 2 min.). During that short timespan, the entire mouse becomes stiff. Can this stifness be taken as a sign of good initial fixation (we post-fix the organs overnight at room temp) or is this stifness not entirely related to fixation ?

Yves

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