Ab25631

Cells were grown on coverslips in 12-well plates (1 × 200 cells/well) overnight and then treated with corresponding chemicals that was performed as previously described. After that, cells were postfixed with 4% PFA for 20 min, then washed with PBS for three times (10 min/time), and then treated with 10% goat or donkey serum diluted in PBS-0.3% Triton X-100 for 30 min at room temperature. Next, cells were incubated with antibodies with P62 (1:500, Sigma, P0067), LC3 (1:200, Santa, sc-376404), LAMP2 (1:200, Abcam, ab25631), LC3 (1:500, Sigma, L7543), SOD1 (1:400, Abcam, ab16831), or Rab7 (1:500, Abcam, ab137029) overnight at 4 °C. The cells were washed with PBS for three times and stained with secondary antibodies, Alexa Fluor 594-conjugated goat anti-rabbit (1:1000, Thermo Fisher, #A-11037) or Alexa Fluor 647-conjugated goat anti-mouse (1:1000, Thermo Fisher; #A-21236), for 1 h at room temperature, then stained nuclei with DAPI Fluoromount-G (Southern Biotech). Finally, imaged the cells by confocal microscopy.

After fixation with 4% PFA, cells were washed twice with PBS containing 0.5% tween 20 (PBS/T; Sigma-Aldrich, P1379) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, T9284) and 0.02% SDS (Acros Organics, 230425000) containing PBS/T for 20 min. To minimize non-specific binding, 0.1 M Glycine in PBS/T was added to the cells after washing followed by blocking in PBS/T containing 1% BSA and 2% FBS. After washing, cells were incubated overnight at 4 °C with various primary antibodies: Rab11 (Thermo Fisher Scientific, 71-5300), Rab5 (Santa Cruz Biotechnology, Sc-46692), EEA-1 (Becton Dickinson, 610457), CD63 (BIO-CONNECT Diagnostics, 11-343-C100), LAMP-1 (Abcam, ab24170), Rab7 (Abcam, ab137029 and ab25245), LAMP2 (Abcam, ab25339 and ab25631), ATP10B (Sigma-Aldrich, HPA034574) or cleaved caspase 3 (Cell Signaling, 9661) diluted according to manufacturer’s recommendations in PBS/T containing 0.1% BSA and 0.2% FBS. Finally, after the samples were washed with PBS-T, cells were incubated with Alexa Fluor dyes (Thermo Fisher Scientific, 1/2000) for 30 min. Nuclei were stained with DAPI (Sigma-Aldrich, D9542-10 mg). Cells were visualized using an LSM780 confocal microscope.