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Another issue surrounding Prolong curing is the creation of a refractive
index gradient within the sample.  We usually cure overnight (~18 hr) in a
fume hood and then seal with nail polish.  In retrospect, we may be
over-accelerating the curing because 3D images of imbedded fluorescent
beads sometimes show an asymmetrical PSF, especially in the Z-plane.  This
indicates that we are not getting a uniform increase in RI during curing--
some parts of the sample may have better exchange with air than other
parts.  Slower curing (say, 2-3 days, not in a hood) might reduce this
problem.  Or, using smaller coverslips.  Or, just using less Prolong during
mounting.

Michael J. Schell
Assistant Professor
Uniformed Services University