Re: Hematoxylin fading (and histoclear)
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From: | "J. A. Kiernan" <jkiernan@julian.uwo.ca> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
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Content-Type: | text/plain; charset="us-ascii" |
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
persistent.
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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