If I remember correctly, heparin (in perfusion fluid?) is used to prevent
clotting problems.  Linda Jenkins has done this, plus some others, any
comments



>Date: Wed, 29 Mar 2000 09:17:16 -0500
>From: Pam Marcum <pmarcum@polysciences.com>
>Subject: RE: perfusion confusion
>To: Roger Moretz <stamptrain@yahoo.com>, Karen Larison
>    <larisonk@uoneuro.uoregon.edu>, HistoNet@Pathology.swmed.edu
>
>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 [mailto:stamptrain@yahoo.com]
>Sent: Wednesday, March 29, 2000 8:22 AM
>To: Karen Larison; HistoNet@Pathology.swmed.edu
>Subject: Re: perfusion confusion
>
>
>Karen:
>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 <larisonk@uoneuro.uoregon.edu>
>wrote:
>> 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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Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303