RE: Perfusion with Tyrode's solution for CNS IHC

From:Cynthia Favara

I just read a relatively old paper comparing various perfusion vs. dunk
fixation in regards to rat brains. Perfusion is the best way to go with a
post fixation in the skull cap for 1-4[if I remember correctly] hours. A
mere tap on the unfixed brain leads to an artifact in the neurons along a
path that presents as a line of neurons with condensed nuclear chromatin and
shrinking. I personally have seen this in many specimens and until now have
never known it was an artifact. In my experience this  is usually  the area
that causes fits with IHC staining.

As to the distress to the investigator and animal. What is your standard for
euthanasia? We are required to take a yearly course here and I am due for
mine but as of last year cervical dislocation is not allowed and this would
be disastrous to the CNS. We deeply anaesthetize the animal and then bleed
them out. 
If the animal is too deeply anaesthetized the bleed is not good and if the
animal is not deeply and you may get a bleeding animal running around. I my
humble opinion and experience if you are proficient with perfusion the time
difference is of no consequence. 

Please remember that everything is compounded as you go through the process
of getting a well stained slide. Is it really worth it to compromise in the
beginning? I like to be assured that I have the best product for myself and
the investigator and prefer to put in the background time and effort when
and if necessary.

If you would like the reference I can get it to you. It is at home.

Best regards and have a great weekend.

Cynthia Favara
903 South 4th Street
Hamilton, MT 59840

-----Original Message-----
From: Andrew Gray []
Sent: Thursday, January 09, 2003 10:42 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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