Please I need some technical advise on multiplex IF for at least 3 proteins:
I am trying to image 3 different endogenous proteins by IF-confocal microscopy, but only have primary antibodies raised in Rabbit and Mouse (i.e., I cannot stain for all 3 proteins without cross-reaction). As a solution, I was thinking to conjugate the primary antibodies to alexa-fluors, however, I have zero experience regarding conjugated Abs and how to go about it. More importantly, I am not sure if this is an optimal solution.
I think, a solution to your problem is multiplexing using tyramides that are conjugated to different fluorochromes.
Such stainings are relatively easy to implement and do not require fiddling around with your primary antibodies and labeling them yourself. Decent starter protocols can be found here:
from Cell Signaling: https://www.cellsignal.com/contents/resources-protocols/fluorescent-multiplex-immunohistochemistry-(mihc)-with-tyramide-signal-amplification-protocol/fluorescent-multiplex-ihc-w-tyramide
OR from Bethyl: https://www.bethyl.com/content/protocol-multiplexing).
I prefer tyramides from Biotium and as secondaries some sort of "polymer" such as the ones from vector Labs (ImPress reagents). The major problem is to define the sequence of your antibodies and making sure you can "cook" off the antibodies after each round of staining.
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