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