Visualizing Mitochondrial Network Morphology

Cells were cultured in chamber-slides for 24 h in the absence or presence of P110. After PBS washes, cells were fixed with 0.3% paraformaldehyde (#P6148, Sigma-Aldrich, St. Louis, MO, USA) and permeabilized with 0.1% triton (#X100, Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated overnight at 4 °C with the antibody against TOM20 (#42406S, Cell Signaling Technology, Danvers, MA, USA). After PBS washes, cells were incubated for 1 h at 25 °C with the Alexa-546-conjugated anti-rabbit antiserum (#A11010, Invitrogen, Waltham, MA, USA) as a secondary antibody. After PBS wash, chamber slides were mounted in mowiol. Immunofluorescence confocal laser scanner microscopy (CLSM) imaging was performed using a laser scanning spectral confocal microscope TCS SP2 AOBS (Leica, Wetzlar, Germany), equipped with Argon ion, He–Ne 543 nm, and He–Ne 633 nm lasers. Images were acquired through a HCX PL APO CS 40×/1.25 oil UV objective and processed with Leica. Images were acquired as single transcellular optical sections.
To evaluate the mitochondrial network shape, fibroblasts were scored depending on the morphology of most of their mitochondrial population as elongated or intermediated/short following the method described in [47 (link)].