[Histonet] IHC theoretical question

wassan alkadhumi w_alkadhumi <@t> yahoo.com
Fri Dec 24 10:30:29 CST 2010

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 
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

Shorsh hospital 
North of Iraq


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