Dmi8 sp8 inverted confocal microscope

Adherent cells on coverslips were fixed in ice‐cold methanol for 10 min at −20°C (for most antibodies) or fixed in 4% PFA for 1 h (for BNIP3‐ANa40 antibody). Fixed cell monolayers were blocked with 2% BSA in PBS for 30 min to reduce non‐specific binding. Cells were then sequentially labelled with diluted primary antibodies and corresponding secondary antibodies for 1 h at room temperature. Coverslips were mounted on glass microscope slides using Fluorescent Mounting Medium (Dako; S3023) or Prolong Diamond Antifade Mountant (Thermo Fisher Scientific; P36965). Images in Figs 2E and EV2E were acquired at room temperature using a DeltaVision Elite inverted microscope system (GE Healthcare) using a ×100/1.4NA Oil PSF Objective from Olympus. Optical sections were processed using the SoftWorx deconvolution algorithm. Images in Fig EV1B and E were acquired using a Leica DMi8 SP8 Inverted confocal microscope equipped with 63× Plan Apochromatic objective. Images in Figs 1D, 2B, 3C, 4F and EV4A were acquired using a Zeiss LSM900 Fast AiryScan2 Confocal microscope with a 63× C‐Plan Apo NA 1.4 oil‐immersion objective. Image deconvolution was performed using ZEN Blue 3D software (version 3.4).