Tomm20

PFA (4%) was used for fixation of primary Hippocampal neurons. Neurons were then permeabilized with 0.5 % Triton and blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature (RT), and incubated at room temperature for 2 hours or overnight at 4°C with the following primary antibodies: MPC1 antibody (1:500, Sigma HPA045119) guinea pig vGLUT1 (1:500, Sigma AB5905); TOMM20 (1:500, Cell Signaling Technology 42406S). Coverslips were then incubated with the following secondary antibodies: anti-rabbit Alexa-fluor568 (1:500, Thermo FisherA21428) and anti-mouse Alexa-fluor488 (1:500, Thermo Fisher A11059) for 1 hour at RT and mounted with anti-fade mounting media (Thermo Fisher P36965), and kept at 4°C until imaged. Confocal images were collected on a Zeiss LSM 880 Confocal Microscope at the Washington University Center for Cellular Imaging which was purchased with support from the Office of Research Infrastructure Programs (ORIP), as part of the NIH Office of the Director under grant OD021629.

The spinal cord sections and PC12 cells fixed with 4% paraformaldehyde (PFA) were subjected to three washes with PBS. Subsequently, the sections were blocked with 10% goat serum in PBS for 1 h at room temperature and then incubated with the following antibodies overnight at 4 °C: NEUN (1:500; ab104224; Abcam), NRF2 (1:100; MA5-42371; Invitrogen), SOD2 (1:200; 66474-1-IG; Proteintech), NF200(1:200; ab254348; Abcam), Tomm20 (1:200; 42,406; Cell Signaling Technology), Drp1 (1:100; 8570; Cell Signaling Technology), and HO1 (1:200; 66743-1-IG; Proteintech). Secondary antibodies, including goat anti-mouse IgG H&L (Alexa Fluor 488, ab6785, Abcam, USA), goat anti-mouse IgG H&L (Alexa Fluor 594, A32727, Thermo, USA), goat anti-rabbit IgG H&L (Alexa Fluor 488, A11070, Thermo, USA), and goat anti-rabbit IgG H&L (Alexa Fluor 594, #8889, CST, USA), were applied for 2 h at room temperature. Finally, the nuclei were stained with Hoechst 33,258 (Beyotime) at room temperature for 15 min. For TUNEL staining, the sections were cultured in a dark and humid environment at 37 °C for 2 h using the TUNEL Apoptosis Assay Kit (C1088, Beyotime) in accordance with the manufacturer's instructions. Images were captured using a Leica SP8 Lightning confocal microscope and light microscope (400× magnification; Nikon, Japan), and eight randomly chosen fields of view were selected for each sample.