Dear histonet members I have a theoretical question concerning IHC, we do HRP method using DAKO materials, first step in immuon staining is to add peroxidase blocking solution to quench endogenus peroxidase. The second step is what am having problem with, we prepare solution from human serum diluted in wash buffer 1:2, as my understanding to why we do this step is to block unwanted proteins in the tissue so that we prevent background staining, but this step is done before adding primary antibody, wont this step block the antigen too since the antigen is a protein too? the other theory i hired is the proteins tend to coagulate together so we add this solution to dispense them, primary antibody then can easily attach to the antigen. we have two histotext books in the hospital and i red the IHC sections, their was really nothing clear about why we do this step and i searched the Internet with no satisfactory answer. As u can see i need help! step #3 primary Ab step#4 link system(secondary Ab against primary Ab (protein) Step #5 chromogen DAB Am doing IHC for a year and 8 months, till now am confused about this step and when i train people am not sure what to say to them about it. help me please Have a great holy day
Wassan Histotechnician Shorsh hospital North of Iraq _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
