ED-1 IHC_ how to remove intense background staining


Dear All,
I am new in the field of immunohistochemistry. So my question may be stupid one.

To analyze renal macrophage infiltration I am using ED-1 Ab from Serotec. My protocol is:
1. deparaffinization with xylene and graded alcohol
2. non-specific protein blocking by 5% normal rabbit serum
3. ED-1 Ab 1:50 dilution in 5% rabbit serum in PBS for overnight at 4 degr C
4. blocking of endogenous peroxidase by 3% H2O2
5. Alkaline phosphatase-labelled polymer of Daco Envision system
6. Chromogen (buffer+fast red+levamisol) of Daco envision system
7. counterstaining with hematoxylen
8. dipping in ammonia water.

But the slides showed intense red background staining with unexpected number of positive cells.

Please give me a suggestion? Do you think that primary Ab (ED-1) is too much concentrated?

Dr Subrata Biswas
PhD student
Nephrology div
UNICAMP, Sao Paulo, Brazil.
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