Fixation and permanent mounting of fluorescent probes after vital labelling of cultured cells

The use of fluorescent probes for the visualization of organelles in living cells and assessment of live/dead cells has an increasing importance in ce ll biology. However, rapid and irreversible morphological changes of labell ed cells (due to the photosensitizing effect of most fluorescent probes) ma ke prolonged observation and detailed analysis of living cells under contin uous excitation difficult. In this study, we describe a method for fixing a nd mounting cultured HeLa and 3T3 cells labelled with acridine orange (lyso somes), rhodamine 123 (mitochondria), Hoechst 33342 (nuclei), and propidium iodide/acridine orange (live/dead HeLa cells subjected to nutrient depriva tion), Fixation is performed with vapours of a commercially available forma ldehyde solution for 0.5-1 min followed by air drying and permanent mountin g in DPX. After this procedure, both the general morphology and selective f luorescent labelling of cells are well preserved. The method of vapour fixa tion and DPX mounting is simple, rapid and reproducible, allowing definitiv e preservation of the fluorescence pattern observed in unfixed cell culture s.