[Histonet] Partially muddy slides

Tony Henwood AnthonyH <@t> chw.edu.au
Tue Nov 16 17:23:53 CST 2010

It might possible be water contamination in the xylene. Try increasing
alcohol dehydration times.
Also re-cyled alcohol is probably around 96% pure. It seems to be not
possible to obtain absolute ethanol by re-cycling, or so I am lead to


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of
histotech <@t> imagesbyhopper.com
Sent: Wednesday, 17 November 2010 4:09 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Partially muddy slides

Hi Histonetters!

We are having an issue in our lab with some inconsistent staining.  It's
an odd situation.  One small biopsy can stain reasonably well over most
of the tissue, but then one area of it is muddy.  This does not affect
all the slides in the same staining rack, nor does it affect all the
small biopsies that were processed in the same processing cycle.
Troubleshooting this has been difficult, at best. It's sort of hit or
miss on when/where the muddy-ness appears.

Here are my thoughts:
** I don't know if this is a processing issue, a staining issue or
perhaps even a collection issue.
** If it affected all small biopsies equally, I would be tempted to
question the processing/fixation.  But it doesn't affect across the
**  If all the staining was muddy, I would look to the stainer, but
again, even within the same slide, we can have clear and muddy areas.
**  The stainer is a Leica XL (about 3 years old),  hooked up to tap
water for the washes.
**  We are using a regressive staining method with Fisher brand Protocol
Harris hematoxylin and Protocol Eosin Y.  We use acid alcohol and
ammonia water for differentiating and bluing.
**  The xylene we use is recycled, but verified for purity.
**  The alcohols are recycled, but the last alcohol prior to the
xylene/coverslipping is "pure", not recycled (for just in case).

Suggestions on what could be causing this issue would be greatly


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