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