RE: Perfusion with Tyrode's solution for CNS IHC

From:"Charles W. Scouten, Ph.D."

Brain cells die within seconds of being deprived of an oxygen supply.
No part of the brain is very long or far from a fresh supply of
oxygenated blood.  Perfusing through the vascular system gets fixative
to every cell in the brain within seconds of entering the chest cavity.

When you extract a rat brain and immerse in fixative, it will take 24 or
more hours for fixative to penetrate all the way in.  Depending on depth
into the brain, the tissue will be in a gradient of states of decay.
One area may stain, another not, based not on antigens present
originally, but on decay in one area and not the other.  

Perfused tissue is much better scientific material than biopsy or
autopsy material.  It's not fun, but is necessary.

Shrinkage due to perfusion can be avoided.  See the following link for a
perfusion apparatus and some references.


Charles W.  Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300  
FAX  314 522 0377

-----Original Message-----
From: Andrew Gray [] 
Sent: Thursday, January 09, 2003 11:42 PM
To: HistoNet Server
Subject: Perfusion with Tyrode's solution for CNS IHC

Dear Histonetters,

I have been attempting some rat brain IHC, and all methods in journal 
articles I have read refer to perfusing with Ca-free Tyrode's solution 
(a paraformaldehyde fixative of some description).   This involves 
anaesthetising the animal (usually with a barbiturate), cutting the 
animal open and pumping fixative into a coronary vessel and draining 
blood, while the animal is still alive (it's not alive for long).   The 
brain is then removed and placed in 4% paraformaldehyde to 'post-
fix'.   I imagine the reason for perfusing is to get fixative to the 
brain as quickly as possible, to minimise changes after death.

This method seems quite time-consuming, labour-intensive and 
potentially distressing to the investigator or animal, not to mention 
the introduction of a CNS-active drug which may affect the results.   
Many parts of the brain are physically distant from blood supply.   
Consequently I have been pondering the value of this type of 
perfusion.   Can any Histonetters comment on this?   I have attempted 
some preliminary experiments without perfusing (just post-fixing), and 
labelling appears good.   Would this deviation from the standard method 
jeopardise the work's chances at publication?

Many thanks,

Andrew Gray
Pharmacist/Pharmacologist/PhD student

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