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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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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the
Purdue University Cytometry Laboratories and distributed free of charge
as an educational service to the cytometry community.
If you have any comments please direct them to
Dr. J. Paul Robinson, Professor & Director,
PUCL, Purdue University, West Lafayette, IN 47907.
Phone: (765)-494-0757;
FAX(765) 494-0517;
Web