Glass chamber slides

The following staining was done according to the protocol specified by Cell Signaling Technology. Briefly, HaCaT and OSCC cells were cultured on glass chamber slides (Eppendorf, Hamburg, Germany) for 3 d and stained with anti-SDHA monoclonal antibody (Cell Signaling Technology, MA, USA) and anti-ACADV polyclonal antibody (Novus Biologicals, CO, USA) and imaged with an inverted fluorescence microscope (Nikon, Japan). The culture medium was removed and the cells were washed 3 times with PBS. Cell samples on slides were blocked with blocking buffer for 60 min at room temperature. The blocking buffer was removed and each diluted primary antibody (anti-ACADV: 2 μg/mL, anti-SDHA: 10 μg/mL) was applied to samples and incubated overnight at 4°C. After incubating, samples were rinsed and incubated for 1-h with secondary antibody before imaging in the Nikon fluorescence microscope.

HaCaT and OSCC cell lines were cultured on Eppendorf glass chamber slides (Hamburg, Germany) for 3 days (d) and imaged, without any stains, using an inverted fluorescence microscope (Eclipse Ts2R, Nikon, Japan). Prior to imaging, cells were washed 3 times with PBS and replaced with non-fluorescent, FluoroBrite DMEM Medium (Thermo Fisher Scientific, MA, USA). Imaging was performed using Nikon’s CFI Plan Fluor 20x objective (NA0.45) and Lumencor’s SOLA light engine. Fluorescence images were captured with a standard GFP filter cube (excitation: 450–490 nm, emission: 500–550 nm, dichroic mirror).