Antifading Reagents For Fluorescence Microscopy

The most frequently recommended additives for mounting media are p-phenylenediamine (PPD), n-propyl gallate (NPG), 1,4-diazobicyclo[2,2,2]-octane (DABCO) and ascorbic acid. These are usually incorporated into a buffer/glycerol solution with pH adjusted for good fluorescence (usually between 8 and 9).

Following are some recipes and references. Please treat these web-based recipes as you would any printed reference: credit the investigators in your reports.

David Spector of Cold Spring Harbor Labs offers a carbonate-buffered medium (rather than phosphate-buffered) using PPD.
John Pringle and others (1989) have used the following for fixed yeast cells.

1. Dissolve 100 mg p-phenylenediamine (Sigma P-6001) in 10 ml PBS, stirring vigorously at room temperature.
2. Adjust to pH 9 if necessary.
3. Add 90 ml glycerol and stir until homogeneous.
4. Store in dark at -20°C for a few months, or at -70°C in aliquots. When solution becomes dark, discard as artifacts will appear.

For live-cell work, it is reported that NPG, DABCO, and ascorbic acid are nontoxic, so they may offer some protection to living cells. Another strategy for preserving live-cell fluorescence is an enzymatic oxygen scaving system. Oxyrase Inc. (Mansfield, OH) is one vendor. For a homemade version see Simon (1995).

References

Giloh, H. and J.W. Sedat (1982). Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science217:1252-1255.
Johnson GD, Nogueira Araujo GM. (1981) A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods43:349-50.
Pringle, J.R., Preston, R.A., Adams, A.E., Stearns, T., Drubin, D.G., Haarer, B.K., Jones, E.W. (1989). Fluorescence Microscopy Methods for Yeast. Meth Cell Biol 31: 357-435.
Pringle, J. R., Adams, A. E. M., Drubin, D. G., and Haarer, B. K. (1991). Immunofluorescence Methods for Yeast. In Methods in Enzymology (C. Guthrie and G. R. Fink, Eds.), Vol. 194, pp. 565-602. Academic Press, San Diego.
Simon, V.R., T.C. Swayne, L.A. Pon. (1995). Actin-Dependent Mitochondrial Motility in Mitotic Yeast and Cell-Free Systems: Identification of A Motor Activity on the Mitochondrial Surface. J Cell Biol 130: 345-54.