[Histonet] RE: DAB haematoxylin counterstain; too purple,

[Rene J Buesa]

To counterstain the IHC sections I always used the regular hematoxylin for 30 seconds only. Always obtained a crisp and weak nuclear staining.
René J.

--- On Thu, 12/15/11, C.M. van der Loos <c.m.vanderloos <@t> amc.uva.nl> wrote:


From: C.M. van der Loos <c.m.vanderloos <@t> amc.uva.nl>
Subject: [Histonet] RE: DAB haematoxylin counterstain; too purple,
To: "'megan.french <@t> mcri.edu.au'" <megan.french <@t> mcri.edu.au>
Cc: "'histonet <@t> lists.utsouthwestern.edu'" <histonet <@t> lists.utsouthwestern.edu>
Date: Thursday, December 15, 2011, 2:55 AM


Hi Megan,
Try to lift the coveslip (soak in xylene for a couple of hours), remove excess of mountant in xylene and then go into alcohol. Dip your slides in alcohol plus hydrochloric acid (95 ml alcohol absolute + 5 ml conc. HCl) for 5 min and then into water. Most of the hematoxylin will be gone.

We use a commercial hematox (Harris) 1:10 diluted in distilled water for 3 min. This ensures and weak to moderate counterstain that is just strong enough to see the tissue elements and not disturbing the IHC signal.

Cheers, Chris

Chris van der Loos
Academic Medical Center
Dept. of Pathology
Amsterdam
The Netherlands

Date: Wed, 14 Dec 2011 14:46:11 +1100
From: "Megan French" <megan.french <@t> mcri.edu.au>
Subject: [Histonet] DAB haematoxylin counterstain;      too purple,
        overpowering IHC
To: <histonet <@t> lists.utsouthwestern.edu>
Content-Type: text/plain;       charset="us-ascii"

Hi all,
I have been working through an immuno using DAB and counterstaining with
harris haematoxylin.

Protocol for counterstain:
5 min h20
1 dip haematoxylin
1 min h20
2 dips Blue in ammonia
1 min h20
Dehydrate, clear, mount

I am finding that my slides are coming out realllly purple and are
overpowering my DAB staining so much so that I can't even see it!

The haematoxylin is new, it was recently changed. I don't think it has
anything to do with the DAB/quenching etc im pretty sure is is haem
related.
Any suggestions would be appreciated!!


Megan French
Surgical Research; Murdoch Childrens Research Institute
E megan.french <@t> mcri.edu.au
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