RE: Uneven Staining

From:"marjorie lehman" (by way of Histonet)

Hi Bob,
These are very simple first steps you might try: since the density of the
epidermis and the dermis are so different I have found that the epi doesn't
flatten as fast as the dermis in the water bath. I allow my sections to float a
bit longer even though they look O.K.. Also, dry the slides standing on edge
and allow them to drain well before you heat them.
Hope this helps. Marge

-----Original Message-----
From: []
Sent:	Wednesday, August 13, 2003 7:20 PM
Subject:	Uneven Staining


I'm posting this for a colleague who doesn't have access to Histonet.  I've
searched Histonet archives and gotten a couple of possible leads, but here are
a few details of this particular problem.  I'm hoping someone has recent
experience with a similar tissue or can help us with a solution to the problem.

Tissue is skin; staining is automated; glass coverslipping is automated;
staining is fairly standard H&E protocol, which has been great for the past
months or so.  And now a "ghost" problem is showing up occasionally, as

There is an occasional area, usually easier to see in the epidermis, which
involves small patches where the H&E staining is much lighter than the rest of
the section.  These areas are fairly small, usually involving about 10 - 20
cells in groups.

I wouldn't call the staining "muddy" or the details obscured.  The nuclear
and cytoplasmic details are still there, but the staining is simply much
than the surrounding tissue.  And I don't see any "streaking" of the staining
running along the length of the slides, so I don't think it's a carryover or
destaining problem.

I'm thinking maybe there is a problem w/ deparaffinization, perhaps with
water getting trapped under the sections in these areas and interfering
with the
first xylene steps.  The slides are normally dried either in a separate
oven or
in the drying oven of the slide stainer.

Does anyone else have thoughts or suggestions on possible causes?  If so,
please post 'em.  Maybe it would be best to post to the list, so we can
the answers via Histosearch (the Histnet archive search engine) when this
problem resurfaces again next year!

Many thanks.

Bob Chiovetti
GTI Microsystems

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