Dic plan apochromat oil immersion objective

Live time series microscopy was performed using a laser scanning microscope (LSM 800; Carl Zeiss GmbH) with a 40×/1,3 DIC Plan-Apochromat Oil-Immersion Objective (Zeiss). Cells were imaged in live cell imaging solution (Invitrogen, A14291DJ) supplemented with 5 mM or 20 mM glucose after starvation or full medium treatment for 24 h. During time series imaging, the cells were kept at 37 °C and 5% CO₂. Airyscan superresolution images were obtained in combination with an electronically switchable illumination and detection module (ESID), with time intervals ranging between 30 and 60 s per position. Projects were processed with ZEN software (ZEISS), whereas the time series stacks were extracted and processed into videos in Fiji. Manual tracking of GFP-WIPI1 puncta within TNTs was performed with the free Fiji Plug-in MTrackJ, where GFP-WIPI1 puncta’s positions were manually selected through each time-lapse frame. Overlay videos were exported displaying tracks of each selected object.

Cells were grown on sterile coverslips and fixed in 4% formaldehyde for 15 min at room temperature, permeabilized, and blocked with PTB buffer (1× PBS containing 0.1% Triton X-100 and 0.1% BSA) for 1 h (Xiong et al., 2015 (link)). The fixed cells were incubated with LAMP1 antibody (Proteintech; 21997-1-AP) at a 1:100 dilution in PTB for 1 h, and secondary antibodies were Alexa-Fluor 568 conjugated donkey anti-rabbit IgG at a 1:1,000 dilution (Invitrogen). The nuclei were stained with 4′,6-Diamidino-2-phenylindole (DAPI) (Sigma). Images of fixed cells were taken using a Zeiss LSM710 Microscope with a 63 × 1.4 DIC Plan-Apochromat oil-immersion objective.

Mixed ventral horn cultures were prepared as previously described [6 (link)–8 (link)]. Briefly, ventral horns from E11.5–13.5 WT and SOD1^G93A mice were dissociated, centrifuged at 380 × g for 5 min, seeded into two-chambered microfluidic devices (Fig. 3A) [7 (link)], and maintained in motor neuron media (Neurobasal (Gibco) with 2% B27 (Gibco), 2% heat-inactivated horse serum, 1% Glutamax (Invitrogen), 24.8 µM β-mercaptoethanol, 10 ng/ml ciliary neurotrophic factor (Peprotech, 450–13), 0.1 ng/ml GDNF (Peprotech, 450–10), 1 ng/ml BDNF (Peprotech, 450–02) and 1 × penicillin streptomycin (Thermo Fisher; 15140122)) at 37 °C and 5% CO₂. After 6 days in vitro (DIV6), 30 nM H_CT-555 and ± 50 ng/ml of BDNF was added to existing media for 45 min, then all media was replaced with fresh MN media containing 20 mM HEPES–NaOH (pH 7.4) ± 50 ng/ml of BDNF for time-lapse microscopy. Live imaging was performed on an inverted LSM780 confocal microscope at 37 °C using a 40x, 1.3 NA DIC Plan-Apochromat oil- immersion objective (Zeiss). Videos were taken at 2 frames/s for > 2.5 min. Videos were manually tracked using TrackMate [54 (link)] to determine endosome track dynamics (Fig. 3). The breakdown of each experimental group can be found in Additional file 2: Table S2.