[Histonet] Partially muddy slides

histotech <@t> imagesbyhopper.com histotech <@t> imagesbyhopper.com
Tue Nov 16 11:09:16 CST 2010

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 board.
**  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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