I am processing tumour tissues for CD3 (ab5690 at 1:100), problem is many tumor cells also takig brown stain
-Heat antigen retrival -20 min
-3 % hydrogen peroxidase after tap water rinse and before protein block
-primary antibody 1:100 dilution 4degree overnight
-secondary antibody goat anti rabbit polyclonal
- DAB time 2 min
kindly give your opinions
with same protocol Thymus and Spleen tissue sections results were good, but not tumor, what would be the reason ?
Are you blocking with normal goat serum prior to primary antibody addition? Its necessary to limit non-specific binding of the secondary antibody.
I think the incubation time is too long and therefore you get unspecific overstaining. The Abcam website tells a incubation time about 10 min at 1:100 for paraffin sections.
Block with 5% goat serum in PBS for 1 hour, check the dilution of ABC complex, DAB and the IgG concentration of your primary antibody. Rinse slides 6 times with PBS for 5min after every step
Thanks to all for your suggestions
I have to not blocked with goat serum prior to primary antibody (procuring it). Little improvement by lowering the incubation time of primary antibody .
hoping getting better after trial with goat serum
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