Plan apochromat oil immersion objective lens

Cells grown on coverslips were washed with PBS and fixed with 4% PFA in PBS for 20 min on ice. After permeabilization with 50 µg/ml digitonin in PBS for 5 min, cells were blocked with 3% BSA in PBS for 30 min, incubated with primary antibodies for 1 h, washed, and then incubated with secondary antibodies for 1 h. Fluorescence microscopy was performed on a DeltaVision Elite (GE Healthcare) equipped with a 60× Plan Apochromat oil-immersion objective lens (NA 1.42; Olympus) and a cooled charge-coupled device camera (CoolSNAP HQ2; Photometrics). All images were deconvoluted using the DeltaVision software (SoftWoRx; Applied Precision Ltd.) to remove out-of-focus material. All images except for those shown in Fig. 2 (E and F) and the insets in Fig. 4 D were z projected by Fiji. For visualization of STX17-positive autophagosomes, cells stably expressing mRuby3-tagged STX17TM were placed on a glass-bottomed dish (617870; Greiner Bio-one) in amino acid–free DMEM (048–33575; Wako) and observed on a DeltaVision Elite. During live-cell imaging, the dish was mounted in a chamber (INUB-ONI-F2; TOKAI HIT) to maintain incubation conditions at 37°C and 5% CO₂. Quantitative analyses were performed on z-projected images using the Fiji and R software.