Re: Cooked tissue (Also under-cooked)

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From:"J. A. Kiernan" <> (by way of histonet)
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  This reply is rather long. If you're only somewhat interested, the
  first paragraph should be enough.

On Fri, 10 Dec 1999, Angeline Meadows wrote:

> I was hoping for any insight you might have in regards to a recent
> here in the lab.  My boss accidentally left our rat brain tissue sections in
> the warm paraffin overnight.  I am getting some really weird labeling and
> to know if this overheating could be the problem.  Has this ever happened to
> any of you or are you aware of problems that could arise?  I'm assuming we
> should just consider them toast - but I wanted check 'the' source before I
> tossed them.

   Opinions vary on this one. I have frequently left specimens (usually
   rat or human brain, decalcified rodent head, or rat gut) in paraffin
   overnight and even over weekends. On a few occasions the temperature
   had gone up to 65C. These tissues never gave any particular difficulty
   with sectioning, and the cooking didn't affect staining by several
   dye and silver methods or immunohistochemical detection of a variety
   of plasma proteins: albumin, coeruloplasmin, IgG etc. It is, of course,
   probable that some antigens will be adversely affected. They all
   respond differently to anything you do to them, but temperatures
   higher than that of molten wax are used for retrieving antigenicity
   that has been masked by fixation in formaldehyde.

   Some people say heat (too long or too hot) has awful effects on
   subsequent cutting and also produces structural artifacts. My specimens
   are always hand-processed, with generous times to ensure complete
   dehydration and removal of alcohol before they go in the 1st of 3
   changes of wax (4 for bigger pieces). I suspect that dehydration
   and alcohol removal may not always be complete when specimens are
   processed by machine (or rapidly by hand). If this is so, there may
   be literal cooking (in hot alcohol/water, which won't mix with wax)
   while the specimens are in the molten wax, and it's easy to believe
   that this would be harmful.

   This topic is discussed in M. Gabe's weighty "Histological Techniques"
   book (Paris: Masson, 1976), which I think of as the last word on
   traditional methodology. Gabe said heat was harmless, but only if all
   water and alcohol had been removed. He was an academic zoologist, so
   he wasn't under any pressure to get those fast results that are
   demanded in a histopathology lab. He did all his own lab work,
   generating four other books and many papers, in the fields of
   carbohydrate histochemistry and neurosecretion. His posthumously
   published "Techniques" book is an English translation of a revised
   version of his 1968 textbook (in French). It is written in an
   extremely readable style that shows how close Gabe was to the
   technology. Its index is abominable, so you have to read lot of
   text to look anything up, but it's time well spent, letting the
   wisdom rub off.

   While on the subject of heat, It's worth pointing out that
   common staining properties of animal tissues, especially with
   "trichrome" stains for connective tissue & cells, are changed
   conspicuously if paraffin processing is carried out with a
   wax that melts at 45C instead of the usual 54-58C. A recent
   paper about this is well worth reading:

   Allison, R. T. and Bryant, D. 1998. Effects of processing
     at 45'C on staining. Biotech. Histochem. 73:128-136.

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

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