Re: formalin substitutes
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | AndreaH@imclone.com |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Wed, 7 Jun 2000 AndreaH@imclone.com wrote:
> What non-formaldeyde based fixers are people using to preserve antigens in
> paraffin sections? I have been using zinc formalin with little improvement
> and am looking for something else. Does anyone used Shandon's Glyo-Fixx?
> Are there tried and true ones out there?
The only reasonable answer to your question is that you should
try some alternatives. This is the only way to find out if the
immunoreactivity of an antigen to a particular serum or
monoclonal is preserved. For preservation of structure, there's
a large body of peer-reviewed literature, much of which has
been summarized in textbooks. The following paragraph makes a
case for trying an extremely simple fixative that's been
around for a long time.
If you are going to cut paraffin sections an acidic and
predominantly coagulant fixative may provide better
antigenic and structural preservation than a neutral buffered
aqueous formaldehyde mixture. A very simple one that's been
around for 149 years is Clarke's fluid: 3 volumes of alcohol
(95% or 100%) and one volume of glacial acetic acid. These
ingredients penetrate rapidly and coagulate proteins and
nucleic acids (respectively) on contact. A 5 mm cube is
fixed in about 6 hours and can then be moved into 95% alcohol.
Don't fix for too long, or there will be some extraction of
RNA and this will spoil any conventional staining method that
makes use of a basic dye.
It's important to know the exact composition of any fixative
you use, and the action of each ingredient. Fixation affects
immunostaining and conventional staining more than any other
preparative event.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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