[Histonet] fixation via perfusion

[Charles Scouten]

See the following link for a discussion of perfusion.  Consider montitoring pressure, not flow rate, see the link to see why, and I agree that the most common error is pressure too low.  The vascular system can clearly handle at least normal cardiac pressure levels, which way exceed typical gravity flow or common perestaltic settings.

http://www.myneurolab.com/myneurolab/mnl_articles.asp?ProductID=471001&idsubcategory=21&idproduct=471001&catdesc=Histology+Equipment&subcatdesc=Sacrifice+Equipment&AddInfoStr=2 


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
http://www.myneurolab.com 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff McAuliffe
Sent: Monday, August 08, 2005 12:22 PM
To: Wisam Barkho
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] fixation via perfusion

Hi Wisam:

    I don't have the exact answer you are looking for but I can provide some direction. This information was probably worked out in the late 1950's and/or early 1960's as perfusion fixation emerged as the way to fix for electron microscopy. I would suggest looking in Hayat's "Principles and Techniques for Electron Microscopy" or Lillie and Fullmer's "Histological Technique and Practical Histochemistry" as places to look for the original references.
    In my experience, most people use too small a canula and/or too low a flow rate for good fixation. The rate of flow you use should approximate the cardiac output of the animal in question. As far as volume goes, I usually use a 3 times the weight of the animal if I am perfusing the whole body, less if you clamp off the lower body and perfuse just the head.
    Good luck!

Geoff

Wisam Barkho wrote:

>Hello,
>
> 
>
>We are doing stereotaxic surgeries on rats in my lab. After 2 weeks, we 
>fixate the brain via perfusion using 4% PFA (after vascular rinse) and 
>then immerse the brains in 4% PFA overnight. My question is where can I 
>find information on pump flow rate and PFA volume to use during fixation? Thanks.
>
> 
>
>Wisam Barkho
>
>daufoi <@t> msn.com
>
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>  
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Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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