Lsm880 airyscan confocal scanning microscope

Cells were seeded onto sterile glass coverslips in 24‐well dishes. Coverslips were washed once with PBS, fixed with 3.7% (w/v) formaldehyde, 200 mM HEPES pH 7.0 for 10 min and washed twice with PBS. Cells were permeabilized with 0.1% triton in PBS for 4 min. After two washes with PBS, samples were blocked by incubation for 30 min in blocking buffer (1% (w/v) BSA/PBS). Coverslips were incubated for 2 h at 37°C with primary antibodies in blocking buffer and washed three times in PBS. Coverslips were then incubated for 1 h at room temperature with Alexafluor coupled secondary antibodies (Life Technologies) in blocking buffer and washed an additional three times in PBS. If needed, nuclei were counterstained with 1 μg/ml Hoechst‐33258 dye (Sigma) for 5 min, and slides were washed twice with PBS and mounted in ProLong Gold (Invitrogen). Observations were made with Nikon Eclipse Ti widefield microscope (Plan Apo Lambda 60× Oil Ph3 DM) or a Zeiss LSM880 Airyscan Confocal Scanning microscope (ZEISS, 63× objective, NA 1.4). Images were processed using ImageJ and Adobe Photoshop Software.

Cells stably expressing the mito-QC mitophagy reporter system (mCherry-GFP-FIS1^101-152) were seeded onto sterile glass coverslips in 24-well dishes. After
treatment, coverslips were washed once with PBS, fixed with 3.7% (w/v)
formaldehyde and 200 mM HEPES pH 7.0 for 10 min, and washed twice
with PBS. After nuclear counterstaining with 1 μg/mL Hoechst-33258
dye, slides were washed and mounted in ProLong Gold (Invitrogen).
Observations were made with a Zeiss LSM880 Airyscan confocal scanning
microscope (ZEISS, 63X objective, NA 1.4). Images were processed using
ImageJ and Adobe Photoshop software.