Immunofluorescence Imaging of LRRK2 and Mitochondria

Fixed BV2 cells were permeablized by 0.1% Triton X-100 in Dulbecco’s phosphate buffered saline (DPBS) for 5 min at room temperature (RT). Coverslips were incubated with a blocking buffer composed of 3% bovine serum albumin and 1% goat serum in DPBS for 1 h. After the blocking step, the mouse monoclonal anti-LRRK2 antibody and rabbit polyclonal anti-Tom20 antibody in blocking buffer were added to cells for 8 h at 4°C followed by the incubation with Alexa Fluoro 488 conjugated anti-mouse (A-11001, Invitrogen) and Texas Red labeled anti-rabbit (T-2767, Invitrogen) secondary antibodies in blocking solution at RT for 2 h. BV2 cells on the inverted coverslip were mounted using ProLong Gold (P36930, Invitrogen) and five images were captured using a Zeiss LS55 confocal microscope operating in the Airyscan mode. BV2 cells expressing GS (n = 10) or transfected cells not expressing GS (n = 4) were selected and their mitochondria (n = 2–20/cell, total 34–107) were analyzed using the Mito-Morphology Macro of Image J software as previously described (Ho et al. 2018 (link)).