As described previously (Lin et al., 2017 (link)), the mitochondria were labeled with MitoTracker Red CMXRos, M7512 (Thermofisher Scientific, USA) for 3 h after the brain slices were treated with LGLO. After an extensive wash, the slices were placed in an airstream incubator at 37°C and imaged by an Olympus inverted confocal microscope using a 60 × 1.3 NA oil immersion objective with 512 × 512-pixel resolution (FV1200, Olympus).
Upon imaging, a total of 5 min with 15 s intervals were imaged for each experiment. The total live imaging time was restricted to 20 min to minimize phototoxic damage. The length, area, and diameter of the axonal mitochondria were measured by the ImageJ program (NIH, USA). The number and mean velocity of motile mitochondria were analyzed by kymographs. Stationary sites in this study were defined as CMXRos-positive profiles that were stationary during a 5-min period. To measure the size of the stationary mitochondria, a pair of image stacks, including all CMXRos-positive profiles of each axon, were obtained at the time periods 0 and 5 min.
Upon imaging, a total of 5 min with 15 s intervals were imaged for each experiment. The total live imaging time was restricted to 20 min to minimize phototoxic damage. The length, area, and diameter of the axonal mitochondria were measured by the ImageJ program (NIH, USA). The number and mean velocity of motile mitochondria were analyzed by kymographs. Stationary sites in this study were defined as CMXRos-positive profiles that were stationary during a 5-min period. To measure the size of the stationary mitochondria, a pair of image stacks, including all CMXRos-positive profiles of each axon, were obtained at the time periods 0 and 5 min.
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