Re: Uneven Staining (by way of Histonet)


 I think you are correct in thinking it is  in deparaffinization. It is
possible the oven is not evenly warmed.  Slides should be in the oven for
at least 15 mins.  If they are microwaving their slides to melt the wax
this can happen also.

Occasionaly I did see this in the lab. There is no particular pattern. I am
sure the water is trapped under the cells in that area, and  have caused
the uneven staining. They may just need to leave their slides in the oven

The other possiblity could be on the staining line. The xylene/xylene
substitute may be dirty, or they possibly could have a bad lot of


Mari Ann Mailhiot BA HT ASCP
Application Specialist
Leica Technical Assistance Center
800 248 0123 x7267
847 236 3063 fax


                      m (by way of             To:
                      Histonet)                cc:

                                               Subject:  Uneven Staining

                      08/13/2003 06:19



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
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

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
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
running along the length of the slides, so I don't think it's a carryover
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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