Goat anti mouse conjugated to alexa fluor 488

For puncta formation, cells were plated on coverslips and transfected with 4 µg/ml of EGFP-LC3 plasmid (Addgene, 11546) 24 h prior to CBR and/or CQ treatment. After 24 h, cells were fixed using 4% paraformaldehyde and mounted using mounting medium (Dako, S3023). For β3-tubulin immunofluorescence, cells were plated on coverslips and after 24 h, cells were fixed using 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS and blocked in 1% bovine serum albumin (BSA) for 30 min at room temperature. After rinsing, the cells were incubated with a mouse anti-β3-tubulin antibody (Santa Cruz, sc-80005), diluted 1:250 in 2% BSA. After overnight incubation at 4 °C, the cells were washed with PBS and incubated in goat anti-mouse conjugated to Alexa Fluor 488 (Invitrogen, A-11001), diluted 1:2000 in 2% BSA. After rinsing, cells were stained for an additional 30 min with TO-PRO3 (Life Technologies, T3605), diluted at 1:1000 in PBS. Cells were then washed and mounted using mounting medium (Dako, S3023). Imaging was performed using Zeiss LSM 510.

For immunohistochemistry experiments, rats were injected with an overdose of 10% chloral hydrate (1000 mg/kg, i.p., Kelong, Chengdu, China) and perfused transcardiacally with physiological saline followed by cold 4% paraformaldehyde (PFA; prepared in 0.1 M of phosphate buffer, pH 7.4). The brains were removed from the skull and stored in 4% PFA at 4 °C for 24 h, then transferred to a 30% sucrose solution at 4 °C for 48 h. 30 μm-thick coronal sections were cut on a freezing microtome (CM3050 S, Leica, Germany) and collected in cold phosphate buffer saline (PBS, 0.01 M, pH 7.4). For immunostaining, each slice was placed in PBST (PBS + 0.3% Triton X-100) with 2% normal bovine serum albumin for 1 h then incubated with primary antibody at 4 °C for 24 h (Mouse Anti-NeuN 1:500, PC213L, Merck). Slices then underwent three wash steps for 10 min each in PBST, followed by 2 h incubation with secondary antibody (Goat anti-mouse conjugated to AlexaFluor488, 1:500, Invitrogen). Slices were washed with PBST (once, 10 min) and incubated for 10 min with Hoechst (1:2000, 861405, Sigma-Aldrich), and then underwent three more wash steps of 10 min each in PBST, followed by mounting and coverslipping on microscope slides. Confocal fluorescence images were acquired on a Carl Zeiss LSM 780 scanning laser microscope (Germany) using a 10 × air objective or a 40 × oil immersion objective.