Mitochondrial network staining via immunofluorescence

Immunofluorescence staining methods have been previously described [18, 22]. Briefly, cells were seeded onto glass coverslips at 250,000 cells/well (in 6-well plates). After overnight attachment, cells were treated with 10 μM TEMS or with 0.25 mM oleic acid for 24 hours. Mitochondrial networks were assessed using antibodies targeting TOM20 (1:100) or TOM70 (1:100) followed by incubation using the appropriate fluorophore-conjugated secondary antibodies. Coverslips were mounted using anti-fade solution (containing DAPI) onto glass slides which were viewed and imaged using the 63X objective (oil immersion) on the PerkinElmer UltraVIEW Confocal spinning disc microscope (CMMB Core Facility, University of South Florida, Tampa, Florida). For quantification, the mitochondrial patterns were categorized according to the following five categories: (1) Tubular elongated; (2) Tubular shortened; (3) Tubular shortened fragmented; (4) Fragmented mitochondria; and (5) Fused not elongated [18]. One hundred cells per sample were assessed and cells were assigned to these five categories.