I am standardizing IHC for pEGFR detection using vector ABC kit, and tried different retrial conditions and antibody concentrations, but I'm unable to get staining. Can anyone suggest any specific modifications that need to be done for phospho Ab detection?
I am using pEGFR tyr 1068 antibody on paraffin -human tissues, tried retrieval with Sodium citrate as well as TE buffer, followed by endogenous blocking with 3% H2O2, 1%serum blocking, primary 1:100 O/N incubation at 4, secondary 1:50 dilution for 45 min RT, ABC for 45 min, DAB development time 10 min.
Are you able to control the tissue fixation? If so, try sampling tissue 2mm slice into cold (4oC) 10% neutral buffered formalin for 2 hours to permeate the tissue then transfer to heated formalin for fixation. Should help preserve phospho sites.
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