Specimen Preparation

Photobleaching

All fluorescent dyes bleach over time upon observation. Oxygen radicals form as a side product of the photochemistry of fluorescence, which react with the dyes and destroy them. Photobleaching is especially problematic with confocal microscopy due to the high intensity of the laser illumination. Inclusion of radical scavengers in the mounting medium can reduce bleaching significantly and prolong the lifetime of fluorescently-labeled samples. Several studies have compared the efficacy of different anti-fade agents, and it is clear that some work better than others^5,6. The two most efficient agents are N-propyl gallate and p-phenylenediamene (PPD). These two have been shown to protect samples far better than other anti-fade agents such as sodium azide, DABCO, or Citifluor. I prefer to use N-propyl gallate however, as PPD discolors over time, producing a brownish precipitate. I have also read anecdotal accounts on various microscopy listservers of PPD actually destroying samples over time.

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