Re: Hematoxylin fading (and histoclear)

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From:RUSS ALLISON <> (by way of histonet)
To:histonet <>
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My co-author Bill Barr also made a study of the degree of fading of
H&E stained sections, as referenced in the relevant chapter of
Cellular Pathology Technique.

He found a reduction in the optical density (OD) following exposure
to sunlight to vary from 14 to 64% at an OD of 536nm and 12 to 51% at
600nm using 22 (Yes twenty-two!) different mounting media.
The 536 nm OD corresponds to the tissue/eosin complex and 600 nm to
haematoxylin/tissue (actually, that ranges from 590 to 610 nm).

The full reference for that piece of work is:- Barr WT.  Effects of
sunlight on stained sections mounted in various media. Stain
Technology (now Biotechnic & Histochemistry) 1970. 45: 9-14.

 However, the point to which Karen refers directly, was reported by
Graham Anderson (NSH Int. Lecturer, 1997), as follows:-"We recently
changed our clearing agent from xylene to Histoclear.....  The
advantage of a non-toxic clearing agent was appealing but, having
used Histoclear for two months, we can record several serious

No copper was demonstrable in liver tissue known to contain copper
bound to protein after processing through Histoclear.  Methyl
green-pyronin stained sections stored in Histoclear for a period of
30 minutes prior to mounting had lost all staining by methyl
green and pyronin was much reduced.  Haematoxylin and eosin stained
sections mounted in resin dissolved in histoclear showed drastically
reduced haematoxylin staining after four days.

Because of these disadvantages and the distinct possiblity of other
adverse staining reactions, we have reverted to xylene as a clearing

Of course, those comments were written fifteen years ago and things
may have moved on a little since.  However, moving from a tried and
tested method to one which may have long term consequences is always
a tricky decision.

Write to us again in fifteen-twenty years.  Meanwhile we will wait
with baited breath for it is a worthwhile experiment.

Happy New Year (and many more to come)
Russ Allison, Wales

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