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

Summary e of fluorescent probes for the visualization of organelles in living cells and assessment of live/dead cells has an increasing importance in cell biology. However, rapid and irreversible morphological changes of labelled cells (due to the photosensitizing effect of most fluorescent probes) make prolonged observation and detailed analysis of living cells under continuous excitation difficult. In this study, we describe a method for fixing and mounting cultured HeLa and 3T3 cells labelled with acridine orange (lysosomes), rhodamine 123 (mitochondria), Hoechst 33342 (nuclei), and propidium iodide/acridine orange (live/dead HeLa cells subjected to nutrient deprivation). Fixation is performed with vapours of a commercially available formaldehyde solution for 0.5–1 min followed by air drying and permanent mounting in DPX. After this procedure, both the general morphology and selective fluorescent labelling of cells are well preserved. The method of vapour fixation and DPX mounting is simple, rapid and reproducible, allowing definitive preservation of the fluorescence pattern observed in unfixed cell cultures.