I´m basically stuck!!! For the last 2months I have been doing standard IHC for the following antibodies: EGF, EGFR, pEGFR.
Although we in the beginning played with the concentrations of primary and secondary to get the optimal protocol, we never get any good results. These last times, we do get staining, but what we see is not in line with what the supervisors were expecting (and therefore questioning my work, which in turn is makng me lose interest in my work and efforts)
Anyone out here that have some experience with antibodies for these proteins/epitopes/antigens and might contribute some usefull advice?
Regarding the protocol itself, it standard and nothing special. No need for antigen masking either.
If the pEGFR doesnt work, can I simply try another generall phospho-tyrosine antibody and try co-label it with some other antibody that I know for sure will be specific to the cell-type I initially wanted to label with pEGFR?
I also wonder wheter it makes any difference whether the EGF antibody is produced in mouse, and the section I stain also is mouse?
Would that makes the staining useless? Will that have an effect at all? I don´t really get that. Can someone please elaborate?
Edited by ProesKhar, 05 October 2013 - 04:29 AM.