Re: [Histonet] brown nickel-DAB staining...

From:Rene J Buesa

  I am not sure if what I am going to tell you will help or not but, there it goes!
  I used 3 drops (about 120 ÁL) of a 2% aq. sol. of nickel sulfate [NiSO4.6H2O] + 4 drops (about 160 ÁL) of 3% H2O2 + 10 mL of PBS + 6 mg of DAB as a second chromogen (permanent) when I did a 2 antigen detection on the same section. 
  I let react x 6 minutes. The first chromogen was the regular DAB and the developed colour with the Ni-DAB was always purplishblue, never brown or black.
  Both colours where present in the 2 different antigen sites, and never as a background.
  To me it seems that you have some dilution to fiddle with.
  Hope this will help after all!
  RenÚ J.

Adam Perry  wrote:
  Has anyone ever performed immunocytochemistry using nickel-DAB as the chromogen in the peroxidase step and gotten brown specific staining (similar to DAB) and the usual purple-black nonspecific background staining in the tissue (characteristic of excessive nickel-DAB reaction product)?

Here's a little more background:

I have site-specifically injected an adeno-associated viral vector that expresses human estrogen receptor into rodent brains that normally express rodent estrogen receptor. When I performed one round of ICC using an antibody that recognizes both human and rodent estrogen receptor, I obtained black (Ni-DAB) stained cell nuclei in regions throughout the brain that normally express estrogen receptor and brown stained nuclei in the area that I injected the viral vector (and presumably contains cells expressing the human estrogen receptor). I then performed ICC using a monoclonal antibody that recognizes human estrogen receptor (rodent specificity hasn't been determined for this antibody yet)...and I only get brown staining in the area I injected with the viral vector- even though I used Ni-DAB as the chromagen (so should have been black)...

I'm thinking maybe the viral expression of the human receptor is so high that there are huge amounts of my protein..and maybe that is somehow affecting the Ni-DAB reaction...but I feel like that's a stretch...has anyone ever gotten similar results (i.e BROWN signal from Ni-DAB with black background staining??)

Any insights would be greatly appreciated....



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