Forwarded: Re: paraformaldehyde questions
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From: | "Karen D. Larison" <LARISONK@UONEURO.uoregon.edu> |
To: | histonet@Pathology.swmed.edu |
Reply-To: | |
Date: | Thu, 22 Apr 1999 11:06:49 -0800 |
Content-Type: | |
Here's another reason to fix at 4 C: According to Fox et al (J Histochem
Cytochem 33, 845-853, 1985), "...tissues fixed at 4 C appeared to have greater
intracellular spaces as though cells had been 'loosened' by the slower rates of
fixation." If I'm doing whole mount staining of embryos, I always fix at 4 C.
Empirically, I have found this yields better staining than RT fix. My
assumption is that the immunogreagents are able to better penetrate the embryo
because of the loosening of intracellular spaces as observed by Fox et al.
Karen Larison - University of Oregon
Date: Wed, 21 Apr 1999 23:19:44 -0400 (EDT)
From: "J. A. Kiernan" <jkiernan@julian.uwo.ca>
Subject: Re: paraformaldehyde questions
To: "Masayuki Miyagishima, MD" <mmiyagis+@pitt.edu>
Cc: histonet@Pathology.swmed.edu
On Wed, 21 Apr 1999, Masayuki Miyagishima, MD asked 2 questions:
> 1) What is the difference between fresh made paraformaldehyde and
> formalin, chemically?
Almost none. As the paraformaldehyde "dissolves" it depolymerizes
and becomes formaldehyde. A formaldehyde solution made by diluting
a stock liquid (37-40%), typically tenfold, also contains a little
methanol (which is included as a stabilizer in the concentrated
solution). An older solution, however made, acquires increasing
concentrations of methanol and formic acid, largely from formaldehyde
molecules reacting with one another. The acidification is
largely taken care of by the buffer or other neutralizing additive
such as calcium acetate, or marble chips. Probably the small
amounts of methanol and formate ions in a working formaldehyde
solution make no difference for ordinary light or electron
microscopy. Conceivably certain enzymes might be adversely
affected. Perhaps someone knows an example.
> 2) Why does the red bible recommend to use 4% paraformaldehyde at 4 C.
> Is it to make the light fixation?
The chemical reactions of formaldehyde fixation are slower than
those of any other fixative agent. Full cross-linking and
structural stabilization take 2 weeks, and you need 4 or 5
days to protect against alcohol, hot wax etc. With brief
fixation and paraffin embedding you see the consequences of
only partial chemical cross-linking of proteins, combined with
partial coagulation by the dehydrating agent. A lower temperature
will slow down the chemical reactions: a rough & ready rule is
half the speed for every 10 deg C drop.
Inadequate formalin fixation can be valuable for several purposes,
including immunohistochemistry when you want plenty of unreacted
stretches in the protein chains. For enzyme histochemistry,
minimal fixation can prevent a cryostat section of an unfixed
object from disintegrating in an enzyme histochemical incubation
medium. In fact you should give as much fixation as you can get
away with. The microanatomy and cytoplasmic details in minimally
fixed cryosections are rather poor by comparison with a plastic
section of something thoroughly fixed in a glutaraldehyde-containing
mixture (glutaraldehyde reacts quickly but penetrates slowly), or
a thin paraffin section following a classical fixative mixture
like Helly's fluid.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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