[Histonet] DAB

[Keri Colwell]

Hello Histonetters,

I am currently trying to work out a new DAB protocol to be used with
IHC on FFPE sections.

I am testing three different protocols, and am having limited success.

The first protocol requires me to make a DAB stock solution of 10mg in
1 ml dH20, which is then filtered and frozen immediately.  This protocol
suggests using DAB tetrahydrochloride XH20, but I am using the free base
of DAB as that is what was on hand.  The free base does not want to go
into solution, but has produced some reaction in my IHC.

My second protocol asks for 40mg DAB in 1.5 ml Tris buffer (0.05M, pH
7.6), to make my stock solution.  I am again using the free base, and
have been stirring since 11:30 am yesterday (MDT) with the result being
a chocolate milk-like substance.

My third protocol requires me to make up the DAB fresh each time I want
to use it. This protocol also asks for the tetrahydrochloride form, but
again I used the free base and did get some reaction.

Does anyone have thoughts on about how to get the free base to go into
solution without oxidizing, or thoughts about free base versus the other
form of DAB?


Thanks!


Keri

Keri Colwell
Laboratory Technologist | Technologiste de laboratoire
TSE and Pathology
Lethbridge Laboratory | Laboratoire de Lethbridge
Canadian Food Inspection Agency | Agence candienne d'inspection des
aliments
Township Road 9-1 | Ch de Canton 9-1
Box 640  | CP 640
Lethbridge, AB T1J 3Z4
E-mail | Courriel: keri.colwell <@t> inspection.gc.ca 
Telephone | Téléphone:  403-382-5500
Facsimile | Télécopieur: 403-382-5583
Government of Canada | Gouvernement du Canada