[Histonet] RE: Making DAB+ in Dako ARKdarker

From:"Fred Underwood"

A few years and brain cells ago I remember incubating slides (after DAB
development) in a dilute silver nitrate solution at 60 degrees C.  The
DAB would blacken nicely, but incubating too long could cause background
staining.  The concentration and times escapes me however.


>>> "C.M. van der Loos"  09/26/03 03:25AM
Hi Ian and Gayle,
There are also non-kit and much cheaper ways to stain DAB any darker 
than the usual brown/yellow. You may add 20 ul of 1% Ni(II)
chloride.6H2O (in dist. water) to 1 ml of a ready-to-use DAB chromogen

solution. The result is a blue/black reaction product. Or, 20 ul of 1%

Co(II)chloride.6H2O to obtain a brown/black precipitate.
Both procedures are described by Hsu and Soban, JHC 30:1079-1082, 1982.

There is also a possibility to post-stain a weakly DAB stained section

with a diluted osmium solution, turning the weakly yellow DAB into 
black. Unfortunately I have lost the exact conditions and reference for

this one.
Have a happy (dark) staining day.

Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands 

>----- Original Message ----- 
>From  Gayle Callis  
>Date  Thu, 25 Sep 2003 08:09:22 -0600 
>To  Ian Montgomery , 
>Subject  [Histonet] Making DAB+ in Dako Ark darker 
>You can make the brown endproduct a very dark, black brown (almost 
>black!) by using the DAKO DAB enhancer.  This works beautifully with 
>their DAB+ that is in ARK kit or you can always do another enhancer to

>turn the product black.  This has been discussed at great lengths in 
>Histonet archives.  Other companies have enhancers ready to use, Zymed

>and Vector, but I am not sure if they are black.   
>Since the kit is a kit, we prefer to use the DAKO enhancing product, 
>pretty much optimized for their DAB+ which (with their enhancer), 
>allowed us to get a murine CD4 diluted out 1:15,000 (0.5mg/ml) and 
>more. This was not an ARK kit method, but we were impressed with the 
>overall chromogen/enhancer. We had to be careful about any residual 
>peroxidase or pseudoperoxidases in tissues when using DAKO DAB 
>enhancer. We used the glucose oxidase endogenous peroxidase method for

>quenching with very clean results.
>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology 
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>>At 01:25 PM 9/25/2003 +0100, you wrote:
>>Although the brown reaction product with Dako Ark looks lovely
>>I'd like a black product. Has anyone used intensifying techniques 
>>with Dako Ark and can they offer any hints and tips.
>>Dr. Ian Montgomery,
>>Graham Kerr Building,
>>Institute of Biomedical& Life Sciences,
>>University of Glasgow,
>>Glasgow G12 8QQ.
>> e-mail: ian.montgomery@bio.gla.ac.uk  

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