Mrc 1024 lasersharp 2000

Immunostaining was performed using the following antibodies: hSOD1 (1:50, Cell Signaling, Danvers, MA, USA); PDI (1:100, ENZO, Farmingdale, NY, USA); ATM (1:100, Abcam, Cambridge, MA, USA); phosphorylated histone protein (pH2A.X) (1:250, Cell signaling, Danvers, MA, USA); and Map2 (1:500, Thermo Scientific, Waltham, MA, USA). Primary cultured neurons were fixed with 4% paraformaldehyde in PBS for 15 min. Blocking was performed using 10% bovine serum albumin (BSA) in PBS-T (0.1% Triton X-100, Sigma, Saint Louis, MI, USA) for 2 h at room temperature. Each primary antibody mixed in blocking solution was incubated with the sample for 2 h and rinsed with PBS-T three times for 15 min. Then, 4’,6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MI, USA) was used for nuclear staining and secondary antibody—Alexa Fluor™ 488 goat anti-rabbit or mouse (1:1000) or Alexa Fluor™ 568 goat anti-mouse or rabbit (1:1000) (Thermo Scientific, Waltham, MA, USA) was used to detect the primary antibody. Confocal and multi-photon microscopy, MRC-1024 LaserSharp 2000 (Bio-Rad, Hercules, CA, USA) was used to obtain the staining images.

All the manipulated plasmids were transfected in the primary cultured neurons and NSC34 cells by using the lipofectamin 2000 reagent (Invitrogen, Carlsbad, CA, USA). Five μg plasmids were mixed with 50 μL of Opti-MEM I medium and 10 μL lipofectamine 2000 was mixed with 50 μL of Opti-MEM I medium. After incubation for 5 min, we combined the plasmid and lipofectamine 2000. The resulting mixture was further incubated for 20 min at room temperature and then transferred into each well containing cells and medium. After incubation in a CO₂ incubator at 37 °C for 12 h, fresh medium was added. The FACS analysis was used to check the transfection efficiency (Figure S1). We fixed both neurons and cells with 4% paraformaldehyde in PBS for 10 min and used both confocal and multi-photon microscopy, MRC-1024 LaserSharp 2000 (Bio-Rad, Hercules, CA, USA) to check protein fluorescence.