RE: perfusion confusion

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From:Pam Marcum <>
To:Roger Moretz <>, Karen Larison <>,
Content-Type:text/plain; charset="iso-8859-1"

We did a large number of perfusions of rats and never used cold fixative.
It was room temperature.  The only person I knew who tried was a grad
student and the perfusion did not go well.  He tried it several times and it
just never worked well.  The brains did not fix well and we had swelling and
burst vasculature due to the cold temperature.  It caused clotting and
problems with processing and cutting.  Pam Marcum

-----Original Message-----
From: Roger Moretz []
Sent: Wednesday, March 29, 2000 8:22 AM
To: Karen Larison;
Subject: Re: perfusion confusion

I don't have any references to the use of cold
fixatives for this purpose, but I can't believe that
perfusion with cold fixative would work that well.
The vasculature will contract upon the initial
exposure to the cold unless a relaxant has been used,
and even then I'm not sure that this will obviate that
problem.  I have always used fixative that is at least
at room temp, and in some instances have run the
perfusion tubing through a warm water
bath--particularly since we were looking at the blood
brain barrier.

Roger Moretz
Dept of Toxicology
Boehringer Ingelheim Pharmaceuticals, Inc.

--- Karen Larison <>
> Histonetters,
> One of the labs here is doing in situ hybridzation
> on developing rat brains.  They
> have been using ice-cold perfusate on these rats as
> they believe that this prevents
> endogenous RNAses from acting.  I have a hard time
> believing this, particularly in
> light of the evidence that paraformaldehyde fixation
> effectively neutralizes
> endogenous RNAses (J Neurosci Meth 85 (1998) pp
> 129-139).  If this is true, why would
> you want to slow the fixation rate by perfusing with
> ice-cold perfusates?
> If anyone knows of any references that would clarify
> this issue or if anyone has had
> some practical experience on perfusion and in situ
> techniques, I'd appreciate hearing
> from you.
> Thanks for your kind advice.
> Karen in Oregon

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