Does any of you try to combine mRNA FISH staining (with DIG-probes and AP antibodies) with protein IHC (with HRP antibodies and TSA signal amplification) on mouse brain sections? They both works, but not when they are combined in one experiment. When IHC is first I can see the signal before the FISH part, but after I can't see any of them. When FISH protocol's first I have the signal from mRNA, but not from my protein. Any ideas why it's not working?
Most FISH protocols use proteases and elevated temperatures to make mRNA more accessible to probes. Most IHC protocols don't make any effort to preserve RNA.
Thus: following IHC, you probably no longer have any intact RNA.
Following ISH, you probably no longer have any intact epitopes.
Note: this will vary from antibody to antibody, while it is unlikely to vary from probe to probe. Some antibodies have such good affinity (or such robust epitopes) that they are almost bomb-proof, and will work on sections even after they've been boiled, digested and repeatedly exposed to stringent washing conditions.
In the sense that this might work at all, ever, it will almost certainly have to be done under ISH conditions, because RNA is just that much more fussy. I have had success with one or two antibodies, but it's rare.
There are kits out there that aim to facilitate this, so it might be worth investigating further, but the take home as far as I'm concerned is: you need a really good antibody, or a really abundant target, or ideally...both.
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