Hello, I am trying to double-stain with Pax 6 and Cleaved Caspase 3. I have looked into the markers but unfortunately, both antibodies are produced in rabbits. I tried Pax 6 and Conjugated Cleaved Caspase 3 in a fixed mouse brain, but it seems like my channels are overlapping. Do you have any suggestions to fix this problem? I used 7%BSA 5%FBS for Pax 6 and 5%BSA 5%FBS for CCC3.
I appreciate your help
Having both the same species as a primary antibody is going be trouble to deal with.
My suggested fix is that you try and search for a new pax6 antibody (https://www.citeab.com/antibodies/search?q=pax6) that is not rabbit host.
Secondly, I suppose you could invest in an antibody labelling kit to try and fluorescently label one of your antibodies directly. I personally have not used these type of kits so if someone who has can give their opinions on using them this could be a viable option.
There is no good way to perform double staining when both your primaries are of the same host. Suppose you can't purchase a mouse Pax6. In that case, using a conjugation kit, the only way to do it is to conjugate your rabbit primary with two different chromophores. This way, you don't need secondaries, as your primary will produce fluorescence by itself, achieving signal separation. In theory, you can try to conjugate your primaries with secondaries in the tube and then use igg to quench the remaining anti-rabbit secondaries, which may or may not work depending if your secondary obstruct the target binding site.
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