C9100 50 camera

Transgenic animals expressing Mito::GFP or mCherry were analyzed at the young adult stage (see table S1). To assess mitochondria density in GABAergic neurons after axotomy, selected axons in transgenic animals expressing Mito::GFP or mCherry were cut using a Micropoint laser from Photonic Instruments (10 pulses, 20 Hz). The axotomized animals were recovered to NGM plates and cultured at 20°C. 6, 12 or 24 hours later, images were acquired as 0.3~0.5 μm z-stacks at room temperature on an UltraVIEW Vex (PerkinElmer) spinning disc confocal microscope (Nikon Ti-E Eclipse inverted scope; Hamamatsu C9100-50 camera) with a 60× CFI Plan Apo numerical aperture (NA) 1.4 oil-immersion objective using Volocity software (Improvision). To score mitochondria density, Mito::mCherry puncta in individual GABAergic axons were counted and then the axon length was measured. The number of mitochondria was normalized by the axon length and converted to density per 100 μm axon length. To compare the volume of Mito::mCherry puncta, Volocity software (Improvision) was used to measure the volume of individual puncta. All animals within each experiment were imaged on the same day with identical conditions including camera gain, exposure settings, and fluorescence filters.

At 16–18 hpf, embryos were anesthetized with Tricaine (Sigma Aldrich), positioned on in a petri dish with a glass bottom (MatTek), and immobilized with low melting agarose (Fisher Scientific). Imaging was performed using a Perkin Elmer spinning disc confocal microscope with a 40× objective lens and a Hamamatsu C9100-50 camera. A Z-series of 1 μm optical slices was taken every 6 minutes for ~1 hour.