Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy.

We have previously reported that the addition of para-phenylenediamine (PPD) to the glycerol used for mounting preparations stained for immunofluorescence with FITC conjugates results in a marked retardation of the fading that occurs during fluorescence microscopy. Studies of the kinetics of this photo-bleaching performed by microfluorimetry and on FITC conjugate in solution and the effects of a range of additives incorporated in the mountant suggest that the fading is due to a destructive reaction with protein of the dye in its excited singlet state, and that retarding additives suppress this. They also indicate the non-participation of singlet oxygen in the process. PPD was found to be the most efficient retarding agent in terms of effective molar concentration of the substances tested, but promising results were obtained with other materials, in particular 1,4-diazobicyclo-(2,2,2)-octane.