Re: mouse perfusion (Long)

From:"J. A. Kiernan"

"Martin, Ronald" wrote:
> My question is - could this perfusion technique be over fixing the tissue? I think she uses formalin (lab made from paraformaldehyde) for the perfusion then continues to fix the tissue in formalin (Fisherbrand) for 4 hours after harvesting it. Do I need to compensate for this procedure by shortening my tissue processing schedule?

Your problem has to be under- rather than 
over-fixation. Over-fixation by a neutral
formaldehyde solution is impossible. The
source of the formaldehyde (formalin or 
paraformaldehyde) does not affect its
actions. Maximum  binding of formaldehyde to
tissue is achieved in 50 hours - see, for
example, a paper by KG Helander 1999 
J. Histotechnol. 22:317-318. Cross-linking
of proteins goes on for much longer than this.
Four hours in any formaldehyde solution is 
not enough time to protect a specimen from
damage by the liquids used for dehydration. 
After only a few hours in formaldehyde, you are
"fixing" the specimen in the first dehydrating 
fluid. If this is 70% alcohol, you'll get fairly 
complete coagulation of proteins and a lot of 
differential shrinkage by the time the specimen 
is in wax. If the first fluid after the formaldehyde 
is 20% alcohol you'll not get much coagulation, and
cells will be damaged by being put into 80% water.
The end result in paraffin sections will be a mess;
nothing like the original micro-architecture of
the organ.

Perfusion starts the fixation evenly, but it
does not speed up the chemical reactions of
formaldehyde, which occur slowly. A coagulant
fixative like alcohol does its stuff as quickly
as it penetrates the tissue, but alcohol alone
is a poor fixative (except for smears of cells
or unfixed sections on slides). To work well
on lumps of tissue alcohol needs to be mixed
with some acetic acid, which opposes the 
shrinking action and also coagulates nucleic

The only reason to fix for a short time in
formaldehyde is when you need minimal chemical
change in the tissue, and sections without
artificial shrinkage spaces around cells,
blood vessels etc. The sections must be cut
either frozen (cryostat or similar) or, if
thick ones are needed, with a vibrating
microtome. Thick frozen or vibratome sections
of minimally fixed brain are used extensively
in neuroscience, for various interesting but
not always good reasons.

It is very easy to damage a mouse's brain
while removing it. Perfusion fixation reduces
the number of "small dark neurons" in cortical
areas touched by scissors or bits of bone.
Everyone doing rodent brain histology should
read a classic paper by Jan Cammermeyer, 1960
in Exp. Neurol. 2:379-405 entitled "Post mortem
origin and mechanism of neuronal hyperchromatosis
and nuclear pyknosis."

John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1

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