Uplansapo 100 na 1.40 oil objective lens

Cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 50 µg/ml digitonin for 10 min or 0.1% Triton X-100 in PBS for 5 min, blocked with 0.1% (w/v) gelatin in PBS for 30 min, and then incubated overnight with primary antibodies against GFP (A-6455, Thermo Fisher Scientific) and LC3B (#2775, Cell Signaling Technology). After washing, cells were incubated with Alexa-Fluor-conjugated goat anti-rabbit-IgG and anti-mouse-IgG secondary antibodies (Thermo Fisher Scientific) for 60 min. Cells were imaged using a confocal laser-scanning microscope (FV1000, Olympus, Inc., Tokyo, Japan) with a UPlanSApo 100× NA 1.40 oil objective lens. z-projection stack images were acquired with z steps of 0.5 µm. SIM images were acquired on a Zeiss ELYRA S.1 microscope equipped with a Plan Apochromat oil-immersion objective (63×, 1.4 NA, Carl Zeiss). Image contrast and brightness were adjusted using Photoshop CS4 (Adobe).

Cells grown on coverslips were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min, blocked with 0.1% (w/v) gelatin (Sigma-Aldrich) in PBS for 30 min, and then incubated overnight with primary antibodies. For Lyso Tracker staining, cells were incubated with Lyso Tracker Deep Red (L12492, Invitrogen) for 2 h. Antibodies against NCoR1 (#5948 S, Cell Signaling Technology; dilution ratio is 1:200), GABARAP (M135-3, Medical and Biological Laboratories, Nagoya, Japan; dilution ratio is 1:200), and Lamp1 (H4A3, Santa Cruz Biotechnology; dilution ratio is 1:200) were used as primary antibodies. After washing, cells were incubated with Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A11008, Thermo Fisher Scientific, Waltham, MA, USA; dilution ratio is 1:1000), and Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A21236, Thermo Fisher Scientific, Waltham, MA, USA; dilution ratio is 1:1000) for 60 min. Cells were imaged using a confocal laser-scanning microscope (Olympus, FV1000) with a UPlanSApo ×100 NA 1.40 oil objective lens. Z-projection stack images were acquired with z steps of 0.5 μm. Image contrast and brightness were adjusted using Photoshop CS4 (Adobe System).