How to eliminate autofluorescence?

Julian F Dye j.dye at ic.ac.uk
Tue May 21 07:20:20 EST 1996


Dear nna

You haven't told us what substratum you have your cells on for viewing.

I am using the technique to examine endothelial cells on glass coverslips
 to look for the intracellular location of a variety of relevant markers,
with FITC and TRITC labelled probes.  Phenol red can't be the problem,
since I don't think I am alone in using it, but I do not have an
autofluorecence problem.

Firstly, are you sure that the fluorescence is truely cellular?  Could
it be the coverslip or slide material? eg plastics such as thermanox are
fluorescent at these wavelengths and are thus unsuitable - use glass.
Could it be a substratum protein? eg cross-linked gelatin coating may
need reducing before culturing.

If the cells themselves are genuinely fluorescent, try borohydride
reduction.  Check the pH of all your reagents.  Check the mountant.
eg Vectorsheild is an excellent one for FITC and TRITC, but it does
fluoresce slightly itself.

Thats all I can think of

Julian Dye
Dept of Anatomy and Cell Biology
Imperial College School of Medicine at St Mary's







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