Re: shrinkage

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From:"J. A. Kiernan" <>
To:"Jennings, Margaret A." <>
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 

> 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

  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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