RE: Quality of Fixation

From:"Charles W. Scouten, Ph.D."

Thanks, to load a picture on the web for histonetters to see, give it a
name and send it to  They will post it so anyone coming to will can see it, then you reference it by name in an email
to the histonet at the usual address.

I claim a better way to perfuse over other ways to perfuse (no
shrinkage, no red blood cells left), the grant reviewer says show that
the fixation is at least as good.  I see it is, but what quantitative or
visual measure (intensity of staining, evenness of staining, etc. can I
use to prove it?  A measure sensitive only to fixation?

So far, the answer is there is not standard test to quantify degree of
fixation.  Some interesting suggestions about proteins stains that are
sensitive to degree of fixation.  I know from experience that HRP
reactions do not work on unfixed tissue, but also that overfixation
reduces HRP sensitivity.


Charles W.  Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300  
FAX  314 522 0377

-----Original Message-----
From: Linda Jenkins [] 
Sent: Friday, March 07, 2003 10:26 PM
Subject: Re: Quality of Fixation

Hi, Charles!

         You stated:

"Is there a standard measure of quality of fixation? A stain that shows
degree of fixation? I don't have a lot of equipment, and don't want to
spend a lot of money, but I have been challenged by a reviewer to show
that my perfusion methods (animal research) results in as good a
fixation as standard methods.
I can see it does, tissue quality is excellent for sectioning and
staining, but how do I quantify or show that? Age it 90 days and
report degree of crumbling? It is 4% formaldehyde fixation."

There are tons of really good references spouting the efficacy of
fixation over "standard methods".  Seems I recall several articles in
Journal of Histotechnology on this same topic.  I am going to send you a
picture that I took of perfused versus non-perfused femurs.  This
really shows the difference!  I think we also have a way to load on a
histonet picture site?  If some kind soul would share that info, I will
also place it on that site.

After reviewing your question I the reviewer questioning
"choice of perfusion method" or "perfusion versus immersion" (there is
comparison between these two methods)?


Linda Jenkins, HT
Clemson University
Dept. of Bioengineering
Clemson, SC 29634-0905

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