Re: shrinkage
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> |
To: | "Jennings, Margaret A." <jennings@mayo.edu> |
Reply-To: | |
Content-Type: | TEXT/PLAIN; charset=US-ASCII |
On Fri, 25 Aug 2000, Jennings, Margaret A. wrote:
> What is the formula for shrinkage when using 10% neutral buffered formalin
> to fix a tissue sample?
Aqueous formaldehyde (buffered or otherwise, with or without salts
to make it isotonic) does not change the volume of a specimen. For
details and references, read J.R.Baker's Principles of Biological
Microtechnique, a great classic in the field of fixation and staining.
> If I start with a tissue a microns x b microns x c
> microns what do I end up with? Is there a time factor for additional shrink?
The shrinkage of a formaldehyde-fixed block occurs during dehydration,
clearing, paraffin embedding and cutting and mounting the sections.
Linear dimensions in the plane of the section are typically 70 to 75%
of the the "fresh" sizes, with much variation among different organs.
If you cut cryostat sections and collect them onto slides or coverslips
they should be approximately life-size in the plane of the slide. The
vertical dimension shrinks with drying. Thick frozen sections, variously
processed before mounting onto slides, may go through large and
uncontrolable size changes. Flattening the section onto the slide with
a brush, prior to air-drying, can cause conspicuous expansion of the
area of a section.
The only way to know the amount of shrinkage is to measure the fresh
specimen and its equivalent dimensions in the stained and mounted
sections.
> Is there also a formula for rate of penetration? Formalin infiltrates y
> microns per t? What about other fixatives any formulas available?
The formula for depth penetrated is useful only for fixatives that
instantly cause a visible change as they diffuse into the specimen.
These are the coagulants, such as alcohol and mercuric chloride.
Baker's book says a lot about this, and provides data not published
elsewhere, as far as I know. Baker explained, with examples, that
in the case of formaldehyde the diffusion formula could not be
used, because the small molecules penetrated specimens rapidly but
reacted quite slowly to produce fixation, and the fixation did
not cause a visible change like that brought about by coagulant
fixatives.
Specimens should be fixed in formaldehyde (especially if neutral
and buffered, which slows down the reactions with protein) for
at least 24 hours to obtain reasonable fixation. For full
formalin fixation you need at least a week. Specimens dehydrated
after only a few hours in formaldehyde are fixed principally by
alcohol. Frozen sections cut after incomplete formaldehyde fixation
are just that: incompletely fixed. Often that's what's wanted,
especially in research work with the CNS.
John A. Kiernan,
Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
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