[Histonet] Going from immmunohistochemistry to immunofluorescence

[Adam .]

Hi all,

I have a few antibodies that I have successfully gotten to work using
immunohistochemistry (primary + biotinylated secondary + SA-HRP + DAB). I am
hoping to move from IHC to immunofluorescence. I tried the same staining
using either a fluorescently conjugated secondary or a biotinylated
secondary and an avidin conjugated fluorophore, but I don't see any staining
at all. I understand the peroxidase chemistry is a lot more sensitive than
fluorescence, so my question is, if I can get IHC to work, should I be able
to get IF to work? If so, how would I go about troubleshooting it? How
important is the flourophore (i.e. Alexa conjugates vs Texas Red vs
Rhodamine).

Yes, I know I could do dual immunohistochemistry, but I like fluorescence
since you can see each stain independently and then merge them
electronically. If anyone has a great suggestion on some chromagens that
work great together, I would welcome those suggestions as well.

Thanks,
Adam