Quantifying ER-Mitochondria Contacts in Alpha-Synuclein

Cells plated on 13-mm-diameter coverslips were transfected with SPLICS [43 (link)] together with empty or WT or mutants α-syn expressing vectors or incubated with TAT α-syn upon the transfection with SPLICS. Fluorescence was analyzed 48–72 h after transfection with a Leica TSC SP5 inverted confocal microscope, using HCX PL APO 63X/numerical aperture 1.40–0.60 upon excitation at 488 nm. Images were acquired by using the Leica AS software. To count ER–mitochondria contacts, a complete z-stack of the cell was acquired every 0.29 µm. Z-stacks were processed using Fiji [55 (link)]. Images were first convolved, and then filtered using the Gaussian blur filter. A 3D reconstruction of the resulting image was obtained using the Volume J plugin (http://bij.isi.uu.nl/vr.htm). A selected face of the 3D rendering was then thresholded and used to count ER–mitochondria contact sites as already described [43 (link),56 (link)].