P35g 1.5 14 cgrd

For electron microscopy (EM), cells were grown on coverslips and fixed in a mixture of 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer for 2–24 h at 4 °C. After post-fixation in 1% osmium tetroxide and 3% uranyl acetate, cells were dehydrated in series of ethanol, embedded in Epon resin and polymerized for 48 h at 60 °C. Ultrathin sections were made using UCT ultramicrotome (Leica Microsystems) and contrasted with 4% uranyl acetate and Reynolds’s lead citrate. Samples were imaged using a FEI Tecnai Spirit G2 transmission electron microscope (FEI Company, Hillsboro, OR) operated at 80 kV. Images were captured with an Eagle 4k HR 200kV CCD camera. For correlative light electron microscopy, cells were grown on gridded glass bottom culture dishes (MatTek; P35G-1.5-14-CGRD) and incubated for 45 min with LysoTracker Red (2 μM) (Thermo Fisher) prior to EM fixation. Fixed cells were imaged on the Nikon A1R laser scanning confocal microscope for LysoTracker staining using z-stacks with step sizes of 0.2 μm as described above, and subsequently processed and imaged for EM as described above.