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
From: Andrew Gray [mailto:Andrew.Gray@vcp.monash.edu.au]
Sent: Thursday, January 09, 2003 11:42 PM
To: HistoNet Server
Subject: Perfusion with Tyrode's solution for CNS IHC
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?
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