[Histonet] another background / non-specific staining problem

[Subratab]

   Dear All

   I am doing immunohistochemistry for 8-OHdG (a DNA marker for oxidative
   stress)  in  rat  kidney  sections  (not perfused) fixed in methacarn.
   Along  with  positive  nuclear  staining, I am getting some background
   staining in some glomerulus and also in some interstitial area.

   This  non-specific  background  is mainly in the areas where red cells
   are abundant (the tissue is not perfused). The same background is also
   present in my negative control slides (absence of primary Ab).

   My protocol is

   1. Deparaffinization

   2. Microwave exposure for 5 min in citrate buffer (10 mM, pH 6)

   3. Horse serum blocking (5%) in PBS for 1 hr at room temp

   4. Primary Ab (mouse monoclonal) against 8-OHdG (N45.1) overnight at 4
   C (diluent 1% BSA and 0.3% triton X 100 in PBS)

   5. Horse anti-mouse secondary Ab 1 hr at room temp

   6. 3% H2O2 in methanol for 10 min.

   7. ABC

   8. DAB

   9. Counter stain with hematoxilin

   10. Dehydration and mounting.


   I  did  some  experiments to identify if this non-specific staining is
   due to endogenous  peroxidase/biotin.


   I  followed above protocol omitting both the primary and secondary Ab.
   I  did  not  get  any  background  stain,  my  slides were clean. So I
   interpreted   that   the   background   is   not   due  to  endogenous
   peroxidase/biotin.  Most probably it is due to non-specific binding of
   secondary Ab with red cell (or other tissue) components.


   Then  I  tried  to  block  non-specific  binding  of  secondary  Ab in
   different  ways  (5%  horse serum, 20% horse serum, 1%, and 5% non-fat
   milk, 1% BSA plus 1% non-fat milk). But I could not able to reduce the
   background. Lastly, I am writing you all for any suggestion.


   Thank you in advance
   Subrata Biswas
   University of Campinas
   SP, Brazil