[Histonet] DAB-Mn histochemistry for superoxide: help to remove background

[Subratab]

Dear histo-experts
I am trying to stain superoxide producing cells on frozen tissue sections of
rat kidney. I am using Brigg’s (and Karnovsky) cytochemical method in
unfixed tissue. The reaction medium contains 1 mg/ml DAB, 1 mM sodium azide
and 0.5 mM MnCl2 in 100mM Tris-HCl (pH 7.2). Incubation time 30 minutes at
37 degree C. (The principle of the reaction is that intracellular superoxide
will oxidize Mn++ to Mn+++; then Mn+++ will in turn cause DAB oxidation and
precipitaion).

MY PROBLEM: I am getting profuse DAB staining throughout the
tubulo-interstitial area (glomerulus is clean). Most probably its from
endogenous peroxidase activity. So I tried to block endo perox by H2O2 (and
also by sodium azide, and H2O2+azide) before incubation with reaction
medium. This way I have removed tubulo-interstitial DAB staining. But I am
not getting any positive stain. I suspect that my blocking step is somehow
damaging (blocking) superoxide producing enzymes as well.

At this stage I need some expert opinion. How can I specifically block
endogenous peroxidase keeping other enzymes intact? Hope to get some
effective ways from histo-experts.

Thanks in advance

Subrata Biswas MD
Lab de Fisiopatologia Renal
Uni of Campinas, SP, Brazil.