Rhodamine Fluorescence After 15-year Storage in Methyl Salicylate

Abstract

Fading of fluorochrome is a significant limitation to fluorescence microscopy. Several anti-fade agents, e.g. n-propyl gallate, are commonly used for glycerol-based mounting media (Longin et al., 1993; Ono et al., 2001). Samples mounted in glycerol must be kept at -20°C for long-term storage to prevent bacterial degradation. In contrast, fluorescent samples cleared and mounted in organic media can be stored indefinitely at room temperature.Methyl salicylate or oil of wintergreen is an excellent clearing agent (refractive index = 1.53), which works well with a variety of fluorochromes. It has a pleasant aroma but is somewhat difficult to work with since it remains liquid after mounting. It was previously reported that shrimp embryos labeled with tubulin antibody and rhodamine-conjugated secondary antibody maintained their fluorescence after six months (Summers et al. 1993). These same samples, stained in November, 1990 and imaged by confocal microscopy for publication in Hertzler and Clark (1992), are still fluorescent after continuous storage in methyl salicylate at room temperature in the dark (Figure 1). The images of 62-cell stage shrimp embryos taken from these 1990 samples were collected with an Olympus Fluoview 300 laser scanning confocal microscope in January, 2006 in the Dept. of Biology, Central Michigan University.