[Histonet] Going from immmunohistochemistry to immunofluorescence

Adam . anonwums1 <@t> gmail.com
Sat Sep 26 18:55:07 CDT 2009

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

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.


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