A1 confocal microscope

STORM was performed using a Nikon A1 confocal microscope with CFI SR Apochomat TIRF ×100 oil objective and Andor Technology iXon3 897 EMCCD camera. Samples were treated as previously described (Huang et al. 2008 (link)). In brief, primary rat cortical neurons were maintained on glass coverslips (1 × 10⁵/ml) for 7 days, then fixed with 3 % PFA and 0.1 % gluteraldehyde for 10 min, and reduced with 0.1 % NaBH₇ for 7 min at RT. Cells were washed three times with 0.1 % sodium cacodylate buffer, and blocking buffer (5 % non-fat milk with 0.2 % Triton) was applied for 20 min at RT. Rabbit anti-Tom20 (1:200; Santa Cruz, CA) and rat anti-tubulin (1:5000; Abcam, MA) were used overnight to label mitochondria and cytoskeleton, respectively. Correspondent fluorescence-conjugated secondary antibody solution (1:2000; Invitrogen, CA) was applied for 1 h at RT. Samples were post-fixed with the same initial fixation solution mentioned above, and coverslips were mounted using imaging buffer with cysteamine (MEA), as described by Nikon, and used immediately. Secondary fluorophores were bleached using 647 and 561 nm lasers until blinking was evident (1–3 min), followed by image recording. Mitochondrial shape and size was measured manually using Nikon AR analysis software (NIS-elements Advanced Research software, Nikon, Japan).