Hello, we are developing a protocol for combining FISH and IHC on Drosophila embryonic brain, using FISH to localize mRNA and IHC (2 antibodies) to mark neuropil and neural tracks. We thought about mixing anti-DIG antibodies with one of the IHC antibodies, but our usual protocol for IHC calls for incubation in antibody for one to two days at 4ºC, and the protocol I found for anti-DIG says to incubate for at least 4 hours at RT. So now we're planning to separate the two processes, but we don't know which one we should do first. Any protocol or suggestions will be appreciated!
If it helps, here are some of the materials we're using:
Anti-DIG-Fluorescein Fab Fragments from Roche
IHC blocking: 100ul normal goat serum + 900ul PBT
IHC protocol: PBT wash (2x20min) -> block (30min) -> 1º staining (O/N) -> PBT wash (3x20min) -> block (30min) -> 2º staining (O/N) -> PBT wash (30min)
I do not think it is a problem using the anti-DIG ON at 4 °C when they state 4 hrs at Rt, as long as the blocking is ok. Always do the FISH first. This can be postfixed with PFA if you are worried about loosing the signal. You could use anti-DIG together with the blocking (then 4 hrs) before the primaries (both primaries together?) or afterwords, together with the other secondaries. Things are also dependent on eventual pre-treatment for the primaries.
Do you want to combine fluorescence (FISH) with using chromogen (IHC) on the antibodies or is everything done in fluorescence? Some chromogens are fluoresce, others might quench signals when expressing at the same location (I had that experience with DAB). I am also a bit worried about weakening of the the FITC signal. It might be necessary to enhance it with an anti-FITC conjugated with Cy2 or Alx488.
Good luck!
Article Combination of Fluorescent in situ Hybridization (FISH) and ...
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