RE: tissue processing (times; also clearing agents)

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From:"J. A. Kiernan" <> (by way of histonet)
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On Thu, 18 Nov 1999, Cynthia Favara wrote:

> John,
> 	Just a question in this regard. I attended a workshop about 4 years
> ago and the information given was that specimens could be over dehydrated,
> and that is why it is necessary to soak blocks. Now what I am apparently
> hearing is that it is not enough dehydration ...

   The presence of water or alcohol in a specimen that is
   supposedly infiltrated with wax is a recognized cause of
   crumbling of tissue when sectioning (see, for example,
   "Cellular Pathology Technique" by Culling, Allison
   and Barr). Some tissues are made unduly hard by alcohol
   and/or xylene, so that they end up harder than the wax.
   Melting ice or cold water is then beneficial because
   (a) the lower temperature makes the wax harder, and
   (b) water soaks into the tissue (especially collagen),
   making it softer but not displacing supporting wax. A
   prolonged soak expands the collagen visibly, and makes
   the tissue bulge out of the faced block.

   Another possible cause of hardening is residual alcohol
   (or worse, water) while the specimen is being heated in
   molten wax.

   Probably there will be conflicting opinions about this,
   but I'd like to suggest that tissue sometimes becomes
   excessively hard because it hasn't been properly fixed
   in formaldehyde (which needs a few days, at least, to
   do its job). The primary fixative thus becomes 50% or
   70% alcohol, which isn't the best.
   I think most people would agree that it is
   easier to cut paraffin sections of objects that have
   been fixed in a coagulant fixative (such as Bouin or
   Carnoy) than in a cross-linker (such as neutral formaldehyde
   - or glutaraldehyde, which is worse). The traditional
   explanation for the difference is that wax molecules
   diffuse more quickly around aggregates of protein molecules
   than through a covalently cross-linked matrix. "Oh let
   us never, never doubt what nobody is sure about." (H.Belloc)

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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