[Histonet] Mucin overstained with Hematoxylin in G.I. specimens

Tony Henwood AnthonyH <@t> chw.edu.au
Sun Nov 4 16:36:30 CST 2007

The mucin staining is usually a function of the Hematoxylin pH. Lowering
the pH (usually by adding acetic acid) can significantly reduce mucin


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of DNon
Sent: Sunday, 4 November 2007 1:42 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mucin overstained with Hematoxylin in G.I. specimens

Hello Histonet, 

     Over the past several weeks our lab has had problems with
hematoxylin overstaining in the mucin of our gastric specimens.  The
degree of hematoxylin overstaining is alarming in the mucin of the
gastric specimens but absent or negligible in other specimens.  One
pathologist reviewing the slides indicated some of the overstained
specimens appeared to be inadequately fixed.  We have addressed that
issue which showed some improvement, but the problem persists.  We are
making changes to increase fixation time (although the specimens now
appear adequately fixed yet retain a large measure of the overstaining),
but any further suggestions for corrective action would be much

    We've been hesitant to increase acid alcohol clearing time because
our other specimens are staining well with the current protocol.

We are using the following reagents and stain times:

-  Surgipath specimen containers pre-filled with 10% neutral buffered
-  Surgipath Gills III Hematoxlyin - 6.5 minutes followed by running
water washes
-  .75% HCL in 70% ETOH for 1 second (1 dip) followed by running water
-  Surgipath Scott's Tap Water for 1 min followed by running water wash

Sakura automated stainer set to agitate ( up and down dipping of racks )
once every 2 seconds, which is the fastest speed available on it.  

Dick Non
Pathology Department
Ocean County Medical Lab
Brick, New Jersey _______________________________________________
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