The defining characteristic of eukaryotic cells is the segregation of critical cellular functions within various membrane bound cellular organelles, including the nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, and mitochondria. Cell biologists therefore have extensively utilized organelle specific counterstains to help identify the localization of specific proteins or other targets of interest in order to garner an understanding of either their potential functions or their effects on the cell. There currently is a wide array of fluorescent dyes and reagents that can be utilized in live and fixed cells to identify organelles, thereby creating challenges in both choosing between the plethora of options and optimizing their use. Here we present a discussion of commonly utilized commercially available organelle dyes and summarize the factors that influence selection of the various dyes for: a given organelle; live versus fixed cellular conditions; adaptation to a specific protocol; spectral multiplexing; or matching excitation/emission spectra to available imaging equipment. Also presented are recommended protocols for a typical example reagent that can be reliably utilized to visualize its target cellular organelle.
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