Immunofluorescence Staining for Mitochondrial Protein

Immunofluorescence staining was performed as previously described [45 (link)]. Cells were grown on coverslips until they reached 60–70% confluence. They were fixed in methanol for 10 minutes at -20°C, washed three times with DPBS (Dulbecco’s Phosphate-Buffered Saline, Thermo Fisher Scientific) and incubated overnight at 4°C with primary antibody Tomm20 (Novus Biologicals, NBP2-67501). After washing three times with DPBS, the specimens were incubated with Goat anti-Rabbit Alexa Flour 488 secondary antibody (Invitrogen, A-27034) for 1 hour at room temperature. All antibodies were diluted in 0.1% BSA in DPBS. Nuclear staining and mounting was performed with Vectashield mounting medium with DAPI (Vector Laboratories, CA, USA). Imaging was performed using a laser scanning confocal microscope (Zeiss LSM 880). All images were taken at 60x magnification with the same laser setting for comparative analysis between the experimental groups.