In other IHC double-label protocols, I see that people reblock the tissue between primary antibody applications. Is this really necessary? Reblocking makes sense if the secondaries of the two primaries are from different hosts, but what if one is plant-based and the other is from an animal?
I'm piloting a double-label where my first antibody is plant-based and fluorescently conjugated. The other primay is not conjugated and it's secondary antibody is from goat. I decided to try blocking with goat serum and a protein-based (non-animal) general blocking solution, respectively. However, I didn't reblock between antibody applications becasue the incubation time of the conjugated primary is only 4 hours. I'm curious to hear what others think about reblocking.
The blocking step is meant to reduce non-specific binding. So you can try with and without and see what happens. If there is increment of non-specific staining in non-reblocked samples, then you must use block solutions every time. On the opposite case, if there is no difference in staining between reblocking and not reblocking, then it is an unnecessary step.
In addition, if your blocking solution is 10% of normal goat serum or whatever protein you are using, I always use ~2% of the same normal goat serum when you prepare the working solutions for primary and secondary antibodies. this helps reducing the non-specific binding.
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