[Histonet] another background / non-specific staining problem
subratab <@t> bdonline.com
Tue Feb 1 11:22:22 CST 2005
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
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.
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
University of Campinas
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