[Histonet] RE: Brigati Stable DAB

[Tony Henwood]

I have found the following very easy to use (an in-house method):

Principle:

The peroxidase enzyme coupled to the ABC Complex is demonstrated using hydrogen peroxide as a substrate and 3,3 diaminobenzidine (DAB) as the electron acceptor.  This causes polymerisation of the DAB resulting in a solvent resistant precipitate.  All histochemical counterstains (except those containing a yellow or brown dye) can be used.
Solutions:

1.	3,3, Diaminobenzidine Tetrahydrochloride Grade II (Sigma D5637).

2.	Tris Buffer-Wash

3.	30% Hydrogen Peroxidase

4.	DAB Stock Solutions 
Caution Irritant
1.	Weigh out 2g DAB in a fume hood
2.	Dissolve in 50ml Tris Buffer-Wash
3.	Label fifty 1ml reagent vials
4.	Aliquot 0.5ml DAB solution in each tube
5.	Freeze and Store at -20oC

5.	DAB Working Solution:
1.	Defrost one vial of DAB Stock.
2.	Add to 50ml Tris Buffer-Wash
3.	Add 50μl Hydrogen Peroxide
4.	Incubate slides for 5-7min until sections appear brown

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
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 Reynolds,Donna M
Sent: Wednesday, 6 July 2011 12:21 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Brigati Stable DAB

I just learned the company making the Brigati Stable DAB is no longer making it. Does anyone know if someone else has been able to pick up this formulation and produce it or are planning to make it. We have been using this for over 20 years and I really hate to change. What DAB are others using that you are happy with?

Donna Reynolds  HT (ASCP),
Chief Histology Lab
Core IHC Lab
Dept. Cancer Biology, SRB 1.660
713-792-8106

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