Antifading embedding media in confocal immunoflourescence microscopy

Fading or bleaching of fluorescence intensity during continuous illumination of stained objects is a serious problem in fluorescence microscopy. Fluorescence intensity as well as bleaching characteristics of dyes are dependent primarily upon physical parameters such as molecular constants (absorption rate and quantum efficiency), excitation energy and brightness (causes photon saturation), and environmental parameters (pH, ions, binding to proteins, etc.) that can strongly influence the properties of fluorochrome molecules. We have studied the effect of various antifading reagents on the behavior of the common dyes fluorescein isothiocyanate (FITC) and phycoerythrin (PE) using immunofluorescent-stained living cells in suspension or membrane-permeabilized dried cells as test systems. As expected, fading cannot be completely eliminated but may be reduced to varying degrees. In our hands, the most efficient antifading reagent for FITC isn-propyl gallate (NPG) dissolved in glycerol. No additive was found to retard fading, but complete dehydration of the cell suspension reduces this effect.