[Histonet] RE: DAB

Tue Mar 17 17:39:05 CDT 2009

I have found the following method easy to use. The DAB Concentrate is aliquoted in small volumes and frozen. It is quite stable for at least a year. The following method is also used by our Haematology department for their myeloperoxidase staining:

Peroxidase
Peroxidase catalyses the transfer of hydrogen atoms from donor (hydrogen peroxide) to acceptor substances. One of the most common acceptor substances is 3,3 diaminobenzidine (DAB) which produces an insoluble, alcohol fast, brown precipitate at the site of the enzyme (Perkins, Henwood & Hewson 1985 “Ultrastructural Myeloperoxidase Localisation:  Applied to Routine Characterisation of Undifferentiated Haemopoletic Malignancies”.  Aust. J. Med. Lab. Sc., 6(3):73-76.). The reaction can be made specific for eosinophils with the addition of sodium azide (Ahlstrom-Emanuelsson et al 2004 Eur Respir J 24:750-757).

Fixation and Sectioning: Air dried unfixed 8µm cryostat sections

Reagents

1.	Phosphate Buffer:
Potassium Dihydrogen Phosphate 	2.622 g 
Sodium Carbonate 	0.778 g
Make up to 100ml. Final pH: 7.2 ± 0.2 at 25°C

2.	DAB Stock Solution
Caution Irritant
1.	Weigh out 1g 3,3, Diaminobenzidine Tetrahydrochloride Grade II (Sigma D5637)  in a fume hood
2.	Dissolve in 25ml Distilled water
3.	Label fifty 1ml reagent vials
4.	Aliquot 0.5ml DAB solution in each tube
5.	Freeze and Store at –20oC

3.	DAB Working Solution:

1.	Defrost one vial of DAB Stock.
2.	Add to 50ml Phosphate buffer in a coplin jar
3.	Add 50μl Hydrogen Peroxide
4.	Mix
5.	Solution remains active for 2 hours

Procedure

1.	Air dry smears (maximum 1hr or alternatively store at -80oC until ready to stain)
2.	Incubate slides for 5-7min until sections appear brown
3.	Wash in water 2-3 minutes.
4.	Counterstain with Haematoxylin
5.	Dehydrate, clear and mount

Results
Peroxidase positive cells		yellow-brown
Nuclei					Blue

Controls

§	Incubate sections in the Incubation medium omitting the hydrogen peroxide
§	To specifically demonstrate eosinophil peroxidase add sodium azide to the incubation medium to a concentration of  0.005%

Regards

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 Swain, Frances L
Sent: Wednesday, 18 March 2009 2:43 AM
To: O'Donnell, Bill; histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] RE: DAB

There was a discussion about the Stable DAB on the histonet a few weeks ago.  I would suggest you search the archieves of the histonet.  The company that has this product was listed at that time.  

Frances L. Swain HT(ASCP) A. A. S.
Special Procedures Technician
Department of Orthopaedic Surgery
Center for Orthopaedic Research
Barton Research Building 2R28
4301 West Markham Street
Little Rock AR 72205
(501) 686-8739 PHONE
(501) 686-8987 FAX
swainfrancesl <@t> uams.edu email
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of O'Donnell, Bill
Sent: Tuesday, March 17, 2009 10:36 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] DAB

Greetings

I recall that years ago Biomeda had a product called Stable DAB. It was a ready-to-use product that was stored in the freezer. Does anyone know if it is still being produced and whom I should contact?

William (Bill) O'Donnell, HT (ASCP) QIHC 
Lead Histologist
Good Samaritan Hospital
10 East 31st Street
Kearney, NE 68847 

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