Ultrastructural Analysis of ER-Mitochondria Interactions

For the ultrastructural study, SV40-transformed H9c2 cells were fixed with 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) at 4 °C for 15 min and with 2% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) buffer at RT for 30 min. After three washes in 0.2 M sodium cacodylate buffer, cells were post-fixed with 2% aqueous osmium tetroxide (Electron Microscopy Sciences) at RT for 1 h, dehydrated in a graded series of ethanol at RT, and embedded in Epon. After polymerization, ultrathin sections (100 nm) were cut on a UC7 (Leica Microsystems) ultramicrotome and collected on 200 mesh grids. Sections were stained with uranyl acetate and lead citrate before observations on a Jeol 1400J EM (Tokyo, Japan) transmission electron microscope equipped with an Orius 600 camera and Digital Micrograph. As previously published [53 (link)], images of ER–mitochondria interfaces were analyzed in a blinded fashion using a custom Image J plugin. The ImageJ plugin for analysis of ER–mitochondrial interfaces in TEM images is available from the update site: http://sites.imagej.net/MitoCare/ (accessed on 8 September 2023).