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> I work with rainbow trout blood cells, and am having problems with
> autofluorescence. We are attempting immunofluorescent staining, but the
> background levels are very high and are making it impossible to
> distinguish specific staining. Examining an unstained blood smear under
> UV light shows overall fluorescence in RBC's and the leukocytes are even
> more strongly fluorescent, particularly around the periphery of the
> cells.
UV and blue-excited autofluorescence is very common (pyridines and
flavins). I haven't done any immuno on fish (all our work has been DNA),
but I would suggest going the other direction; use a HeNe laser and a
red-excited fluorochrome like APC. Good luck!
Steve
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Steve G. Hilliard : "I find sometimes it's easy to be
Cell Analysis Facility : myself. Sometimes it's better to
University of Georgia : be somebody else!"
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