[Histonet] DAB black/brown

[Rene J Buesa]

Peter:
  Just bear with me if I review with you my protocol:
   
  For a second chromogen (double staining) I used:
  DAB --- 6 mg, dissolved in 10 mL of PBS to which I added 160µL of 3% H2O2
  To this mixture I added 120µL of a 16,2% aq. solution of nickel sulfate hexahydrated.
  I let the solution to act for 6 minutes. Results = BLUE-PURPLISH, but I never got to a BLACK counterstain, which makes me think that you are using too much NiSO4.6H2O 
  In all reality, I think that blue versus brown is a better contrast for a double stain.
   
  If you are doing it exactly this way I do not know what to tell you, but I think that if in the same slide you are getting different hues in different areas it may be due to unequal distribution of the solution or an uneven solution distributed evenly.
  Hope this will help you!
  René J.

Boor Peter <boor <@t> email.cz> wrote:
  Can anybody help?

For our IH we're using DAB with Ni chloride to get black color (especially
for double stains with AEC). 
In the last period however we get also brown coloured stainings and even in
one run we get some slides with brown some with black color (very bad for
our double stains).
We used new DAB, made fresh Ni chloride solution and H2O2 but nothing helped
till now...we now even got some slides stained half black half brown...
This is especially bad for our double stainings (it's very hard to
distinguish brown from red).

Where could be the problem? 

Thanks a lot for any suggestions!

Peter Boor


Peter Boor

University Clinic of Aachen, Dept. of Nephrology and Immunology, Pauwelsstr.
30; 52074 Aachen; Germany 

Home: Kullenhofstr. 54A, App. 549; 52074 Aachen; Germany

Mob.: 0049 (0) 177 2345 163

Lab: 0049 (0) 241 80 89670

Fax: 0049 (0) 241 80 82446

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