[Histonet] RE: Making DAB+ in Dako ARKdarker

[C.M. van der Loos]

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 <gcallis <@t> montana.edu> 
>Date  Thu, 25 Sep 2003 08:09:22 -0600 
>To  Ian Montgomery <ian.montgomery <@t> bio.gla.ac.uk>, 
>Histonet <@t> lists.utsouthwestern.edu 
>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
>MT,HT,HTL(ASCP)
>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.
>>Ian.
>>Dr. Ian Montgomery,
>>Histotechnology,
>>Graham Kerr Building,
>>Institute of Biomedical& Life Sciences,
>>University of Glasgow,
>>Glasgow G12 8QQ.
>> e-mail: ian.montgomery <@t> bio.gla.ac.uk