[Histonet] DAB

Tony Henwood AnthonyH <@t> chw.edu.au
Wed Jul 7 19:15:02 CDT 2010


For the free base of DAB you will need to dissolve it in N,N dimethyl formamide. The free base is poorly soluble in aqueous solutions.
Substitute the formamide for the distilled water:

1.	Weigh out 2g DAB in a fume hood
2.	Dissolve in 10ml N,N dimethyl formamide
3.	Label fifty 1ml reagent vials
4.	Aliquot 0.1ml DAB solution in each tube
5.	Freeze and Store at -20oC

For use:

Add 0.1ml DAB solution to 50ml buffer and add 50 microlitres of 30% hydrogen peroxide.

I prefer to use the DAB tetrahydrochloride


Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead 
Cnr Hawkesbury Road and Hainsworth Street, Westmead 
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Keri Colwell
Sent: Thursday, 8 July 2010 7:10 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] DAB

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?



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

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