Here is my thinking: In theory, antibodies have higher affinity for their antigens/epitopes than the proteins in the serum used for blocking. Why not have them compete for the binding sight at the same time? It is common for antibodies to be diluted in the blocking buffer itself in western blots. So why not ICC?

I have already attempted this and do not observe any significant differences from blocking in a separate step. This cuts out some time and makes the protocol more efficient if there are no issues to be seen.

I dilute primary conjugated antibodies in Blocking Buffer which is normally 10% serum. I did an experiment using 10%, 5%, 2%, and 1% serum Blocking Buffer. I incubated for 1 hour at RT, then placed in ~4oC overnight. Then washed and imaged following morning. The fluorescence, background, and staining does not seem any different from previous experiments where I used Blocking Buffer in a separate step and then diluted the antibodies in Dilution Buffer(which has 1% serum) and incubated after blocking.

The only thing I am worried about is if any of this has an effect on non-specific binding?

What is the reason for why this is not typically performed in ICC?

Thank you.