[Histonet] DAB: old-fashioned style!

James L Burchette burch007 <@t> mc.duke.edu
Fri Aug 29 12:45:25 CDT 2008

Diaminobenzidine (DAB)*

DAB Stock Concentrate, 20 mg/ml
        In 50 ml of room temperature 0.05 M Tris Buffer, dissolve 1.0 g 
3,3' Diaminobenzidine powder (Sigma D5637).
        Filter and aliquot in desired amounts.  Freeze for future use.
        Keeps indefinitely at -85 C  Once thawed, use within 2-3 hours. 

*Note:  DAB is a suspected carcinogen, use a hood, wear gloves, eye 
protection and mask when using DAB powder.
        Wear gloves when handling DAB stock liquid. 

Working DAB, 0.5 mg/ml
        Thaw DAB stock aliquot, add to the appropriate volume of room 
temperature 0.05 M Tris buffer and add the appropriate amount of 0.6% H2O2 
for the desired total volume of working DAB 
        DAB Stock Volumes       DAB Concentration       add     0.6% H2O2 
QS with Tris to final volume of:
        0.5 ml  =       10 mg   +       0.3 ml          20 ml
        1.25 ml =       25 mg   +       0.75 ml         50 ml
        2.5 ml  =       50 mg   +       1.5 ml          100 ml

Reaction time is usually 3-5 minutes.

0.05M Tris buffer, pH 7.5-7.6
        1.39 g Trizma base
        6.06 g Trizma HCl
        QS to 1000 ml with D H20

0.6% Hydrogen Peroxide
        To 98 ml of DH2O add 2 ml of 30% H2O2.  Wear gloves and eye 
protection when using 30% H2O2.  Use as needed. 
        Has a 4 month shelf life.  Store at 4oC. 

Imidazole Tris Buffer, 0.05M
Used for DAB enhancement
1.39 g Trizma base
6.06 g Trizma HCl
3.4 g Imidazole, FW 68.08
Mix in 950 ml, adjust pH to 7.6, QS to 1000 ml
Add your above stock DAB aliquot and H202 to this buffer for the desired 
final volume if you like an enhanced DAB reaction.
You can also use 1% copper sulfate as an enhancement for 15-30 seconds 
after rinsing the DAB from your slides.
Add 200 ul of 1% cobalt chloride to 50 ml of working DAB solution for a 
blue DAB reaction. I have never tried a post DAB enhancement with cobalt 
chloride, but I think it should work.


Jim Burchette, HT(ASCP) QIHC
"A simple histotech from a little country hospital in North Carolina"

Kim Merriam <kmerriam2003 <@t> yahoo.com> 
Sent by: histonet-bounces <@t> lists.utsouthwestern.edu
08/29/2008 01:08 PM

Histonet <histonet <@t> lists.utsouthwestern.edu>

[Histonet] DAB: old-fashioned style!

Hi All,

It's been many moons since I had to make DAB by hand and I have lost my 
recipe.  Can anyone let me know how to make DAB from powder and H2O2?

 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 

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