looking for: direct immunocytochemistry protocol - (Nov/15/2006 )
I'm looking for a good protocol for use with biotin-coupled primary antibodies. (My guess is that you just simply omit the secondary antibody and after the binding of the first you just proceed to the streptavidin-step. But I haven't done any ICC or IHC before.)
Are there anything I have to be on a lookout for in direct ICC? I'm worried that the background will be higher due to the lack of secondary Ab. How do you quantify the data anyway? CellProfiler?
(The problem is that I need to compare integrin-levels on different cells. I was advised to use ICC because there was no monoclonal Ab avaliable from the preferred company. Now they ordered this biotin-conjugated monoclonal stuff -do you think it would be best to return to the original idea and use ELISA?)
Thank you very much.
Using a biotin conjugated primary and streptavidin-HRP enzyme (you had it right) will actually give you less background (unless there is endogenous biotin in your cells...). I am not familiar with ICC though. My experience is with IHC.