1.32 na numerical aperture objective

HeLa cells were grown on 35-mm plates (MatTek), fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.2% Triton-PBS or fixed in Methanol/Acetone for 5 min depending on the antibodies used. After blocking for 1 h in 5% BSA in PBS-0.05% Tween 20, the plates were incubated 1 h with the appropriated antibody at 4°C. Cells were imaged on a Leica DMIRBE inverted microscope equipped with a TCS SP5 confocal scanner and a 63× 1.32 NA (Numerical Aperture) objective (Leica Microsystems). Deconvolution of z-stacks was achieved with Huygens software (Scientific Volume Imaging) and 3D reconstruction of deconvoluated images with the Imaris software (Bitplane). Imaris wizard to detect spots was used to quantify 2XFYVE-GFP positive vesicles (threshold of 0.8 µm diameter and intensity 1.5 fold higher than background). Live cell imaging was performed on the same microscope but a 37°C chamber, an objective heater and a 5% CO₂ chamber were added to the system. Cells were placed in phenol red free media the day before imaging them.