I will be carrying out my first tissue array screen for expression of our protein of interest by immunofluorescence on commercial FFPE sections. I have tested the primary antibody in FFPE adipose tissue along with "secondary only" control and got satisfactory results. Do I need to run secondary only controls for the whole tissue array? Thanks for any comments!
Hi Catherine, I'd strongly recomend to run secondary only controls every time.
Maybe in the majority of the cases everything is going to work well, but if it doesn't You'll never know why and that's extra headaches you can avoid early.
To be blunt: secondary only controls are of limited value. This type of control tests what generally is the more reliable component of your stainings: the secondary antibodies. If the secondary only is not beyond doubt, then you have to switch to another source for your secondary. The problem in IF and IHC stainings is the primary antibody. How much evidence do you have accumulated to say that your stainings with the primary antibody are fine and the results you get represent well the protein expression of your gene of interest? How specific is your antibody? These questions can be very hard to answer for poorly characterized proteins and genes.
The commercial TMA slides can be expensive. I would not waste a slide on a secondary only control. I would combine my antibody for your gene of interest with Ki67 or something else that is well expressed in well-defined cells. Every core that does not stain well for, e.g. Ki67 (low intensity, weird pattern etc.), I would eliminate from the analysis. people make such a big fuzz about normalization in qRT-PCR experiments, Westerns etc., but when it comes to IF/IHC most people don't do the minimum quality control for TMAs: does the individual core allow for good staining based on a reference antibody staining. I would do a double IF with a trusted antibody and your antibody of interest. If the trusted antibody does give dubious results on some cores, carefully evaluated these cores and exclude them from the analysis if you deem them "bad".
This does not solve the problem with the reliability of the primary antibody. Before you go on a TMA, I would perform a rigorous evaluation of it, in case you have doubts about the specificity of the primary. You seem to be happy with the secondary system, so I would focus on the primary. This is generally the weakest link of your staining procedure.
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