[Histonet] DAB haematoxylin counterstain; too purple, overpowering IHC

[Maxim Peshkov]

Megan,
Your contersain DAB protocol has two weak point:
1- ammonia water. I used instead ammonia wash buffer
(Tris-EDTA). It is very good and soft for IHC sections.
2- your blueing time is very short.
Please try after 20-30 sec in hematoxyline dip for 1-3
mins slides into wash buffer, then finish as usual and
you will see clear and crisp counterstain.
If hematoxyline will be to much, then you can
differentiate it in 0.25% HCl 5-10-15 dips, then wash
buffer.
Hope this help.
Maxim Peshkov,
Russia,
Taganrog.

> From: "Megan French" <megan.french <@t> mcri.edu.au>
> Subject: [Histonet] DAB haematoxylin counterstain;   too purple,
>         overpowering IHC
> To: <histonet <@t> lists.utsouthwestern.edu>
> Message-ID:
> <DE098B84182F9B4F966EE97D926888D9B48C31 <@t> murmx.mcri.edu.au>
> 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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