Re: Manual H&E staining problem

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From:Mick Rentsch <> (by way of histonet)
To:histonet <>
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Dear Pam,
firstly I assume your nuclear stain is working and the areas where the eosin
fails the nuclei are stained. While it is most likely a failure to properly
dewax and bring to water as indicated by Rande Kline, good Eosin staining
also relies on pH and performs best at or about 5.3 while at pH 8 you won't
get any staining at all. If you are using an alcoholic eosin, one of my
trainees once made it up in Methanol instead of Ethanol, and we got
virtually no stain retention.
Some years ago, we followed the directions for our Honeywell GLS360 stainer
(now Shandon, also known as Lerner and originally as SKI) and after mounting
our sections and giving them an initial drain, put them straight on the
Machine; we then had this problem you describe  being random on the same
slide and the Haematoxylin was also affected, and different areas on
different slides: the problem was very simple, we needed to thoroughly dry
our slides for at lest fifteen minutes in a forced hot air dryer, then put
them on- results no more patchy staining. Cause:- trapped water denied
access of the Xylene to the wax in the section.
Moral of the story:- dry your sections well, for as long as is practical in
your work situation.
Regards Mike Rentsch (Downunder)
-----Original Message-----
From: Pam Plumlee <>
To: '' <>
Date: Friday, 13 November 1998 5:17
Subject: Manual H&E staining problem

>Histonetters: I do manual H&E staining on human and animal tissue.
>Lately some sections or parts of sections are not taking up the Eosin.
>This problem is very intermittent, but very noticeable on slide review.
>Sometimes the first level on a slide will stain great and on the third
>level the whole right half of the section will not stain.  I've tried
>drying the slides longer, alternating the side holder from side to side,
>to make sure the paraffin melts correctly.  Any ideas?  Thanks in

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