[jinli999 (OP)]

It is known that in the procedure of Immunofluorescence, you need to add blocking solution to get clear results. My question is:

Using goat serum as an example to block non-specific binding sites, do blocking solution(goat serum) also blocks those specific sites that primary antibodies bind to ? if the specific sites were blocked by blocking solution ,then how do primary antibodies bind to them? Or just some of them might be blocked, leaving some others unoccupied?  Then how do you tell whether the secondary antibodies bound  to the first antibodies or non-specific binding sites?

Would it possibly happen when most of specific binding sites are blocked by blocking solution, leaving less specific binding sites than non-specific binding sites generating false positive results?

[chris]

I think you are thinking about this the wrong way around. The purpose of blocking solution is to prevent non-specific attachment of the antibody to the target. By adding blocking solution any sites on the target that non-specifically bind proteins will become saturated by the epitopes in the block solution. Then, when you add the antibody, because these sites that randomly bind proteins are blocked, the antibody then binds in a highly specific manner only to the sites that it recognises, rather than the other way around.