Fitc labeled goat anti rabbit antibody and rhodamine labeled goat anti mouse antibody

Cells cultured on coverslips were washed with cold PBS, fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100. After incubation with 2% normal goat serum (to block non-specific staining), fixed cells were incubated overnight at 4 °C with antibodies against Tom20 (1:500, Santa Cruz Biotechnology, USA). Cells were washed with PBS and incubated for 60 min with FITC-labeled goat anti-rabbit antibody and rhodamine-labeled goat anti-mouse antibody (1:500, Invitrogen, USA), followed by incubation with Hoechst dye (1:10000, Invitrogen, USA) for 10 min. Coverslips were mounted and slides were imaged by confocal microscopy (Olympus, Fluoview FV100). To determine mitochondrial superoxide production in cultures, cells were incubated with 5 μM MitoSOX^™ red mitochondrial superoxide indicator (Invitrogen) for 10 min at 37 °C. To measure the membrane potential of mitochondria in cultures, cells were incubated with 0.25 μM Tetramethylrhodamine (TMRM) (Invitrogen) for 20 min at 37 °C. The staining was imaged by microscope, and quantification was carried out using NIH Image J software.