Re: Hematoxylin fading (and histoclear)

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From:"J. A. Kiernan" <> (by way of histonet)
To:histonet <>
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On Mon, 4 Jan 1999, Karen S Pawlowski wrote:

> My boss just noticed in the 1985 edition of Cellular Pathology Technique,
> Culling, Allison and Barr eds., that food oil derivatives, such as
> histoclear, will cause "haematoxylin" staining to rapidly fade.  Is this
> true?  I have been having problems with hematoxylin fading for quite some
> time and I use histoclear in my paramount all the time. 'Tho I began (8
> years ago) by using xylene, and I think those faded just as much.
> If histoclear does cause the stain to fade, are there any other
> substitutes for xylene that would work?

  R. D. Lillie made extensive studies of mounting and fading
  in the 1940s and 1950s (reported in Ch. 5 of his
  "Histopathologic Technic" book, with references to papers
  in Stain Technol).

  Fading is typically due to either acidity or chemical reduction.
  Canada balsam is acidic (abietic acid is a major component)
  but this doesn't matter - the acid can't ionize in the
  absence of water. Alum-haematoxylin stains keep for many years
  in Canada balsam though an eosin counterstain may fade a little
  over the course of 2 years. (This must be from reducing agents
  in the medium.) Easily reduced colours fade in Balsam (e.g.
  Prussian blue, acid fuchsine) and are more stable in synthetic
  media. Easily oxidized colours (notably cobalt sulphide) are
  well preserved in Canada balsam but fade in a few months in
  most synthetic resins. (You can revive CoS by removing the
  coverslip and exposing to ammonium sulphide again.)

  Limonene (I think this is what histoclear is) is a reducing
  agent - two non-conjugated double bonds in the molecule - so
  its presence in a mounting medium is undesirable. Some is
  bound to persist, just as some xylene persists when the
  mounting medium dries. The boiling point of limonene (176C) is
  a bit higher than xylene (140C), so it might be a bit more

  If your eosin faded equally after xylene or limonene the
  mounting medium is the probable culprit. Try a wholly synthetic
  (polystyrene or methacrylate) medium rather than a natural
  or semi-synthetic one. Synthetic media are the best bet for
  pretty well everything except cobalt sulphide (from ATPase
  histochemistry etc). The blue component (alum-haematein) of
  an H & E stain is very resilient and should not be affected
  by clearing agents and mounting media. It keeps for decades
  (perhaps for ever). If yours is fading, something acidic and
  probably also some alcohol must be passing through into
  the mounted preparations. This is surely unlikely to occur
  on a regular basis!

  Hope this helps. I'm sure I haven't thought of everything,
  and have no personal experience with histoclear/limonene,
  but you should be getting lots of other suggestions too.

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

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