Re: Perfusion with Tyrode's solution for CNS IHC

From:"J. A. Kiernan"

For at least 50 years, perfusion has been the 
standard way to fix the CNS for research purposes.
For the wash-out it isn't necessary to use
Tyrode's solution. simple saline or PBS is fine.
A little heparin may be added (anticoagulant)
and so may a little sodium nitrite (vasodilator).
You need to get the vessels free of blood so
that they won't clog when the fixative arrives.

Remember that perfusion speeds up only the
delivery of the formaldehyde, not its chemical
action on the tissue. Perfusion followed  by
brief immersion (or simply leaving the corpse
in a fridge for a few hours) is a way of
obtaining minimal fixation of the brain, when
that is desired - usually to be followed by 
cryoprotection in sucrose and cutting frozen 
sections. If you want the brain to be properly
stabilized by fixation you must leave it
immersed in formaldehyde for a few days, at
least, after perfusing. 

Perfusion fixation is, as you point out,
labour-intensive. It isn't distressing, however,
to either the investigator or the animal.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Andrew Gray wrote:
> 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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