Lsm 900 airyscan 2 confocal microscope

For immunofluorescence staining, SCC011 cells were grown on cover slips until the reaching of 80% of confluence, fixed with 100% cold methanol on ice, and permeabilized in 0.1% Triton X-100 for 15 min. After permeabilization, cells were blocked with 0.3% BSA/PBS and washed in PBS. Next, cells were incubated with primary γ-H2AX (anti-phospho-Histone H2A.X (Ser 139) clone JBW301, Cat. 05-636, Millipore, Burlington, Massachusetts, USA) and RAD51 (Rad51 (H-92), cod. sc-8349, Santa Cruz Biotechnology) antibodies (for antibodies dilutions see Supplementary Data 3), O/N at 4 °C. Secondary antibody incubation was carried out at room temperature for 1 h, followed by washing in 0.2% Tween/PBS. Images were acquired with a ZEISS LSM 900 Airyscan 2 confocal microscope. For 8-oxo-dG assay, the immunofluorescence staining was performed according to the manufacturer’s instructions, by using the anti-8-oxo-dG antibody (TREVINGEN, cod. 4354-MC-050). The measure of the co-localization of 8-oxo-dG/DAPI signals was performed by counting the number of nuclei in a field. Briefly, after counting the number of nuclei in each field, we determine the number of foci, normalizing for the number of nuclei in each field. The foci in each region were defined by the nuclear staining overlap.

Images for luminance quantification were taken using a LEICA M205 FCA at 1X. A 500 ms exposure was used to measure the native fluorescent luminance from GFP based on the versatile dynamic range revealed in an exposure series analysis of 100, 500, and 1,000 ms (Supplementary Figure S1). GFP luminance analysis of striatum was performed on ImageJ with an ROI restricted to the expression in striatum. A Zeiss LSM 900 Airyscan 2 confocal microscope was used to collect images for colocalization of GFP with cell markers labeled with immunohistochemistry. For quantification of colocalization, cell counts of images taken with 20X and 63X objectives were quantified using ImageJ.

For immunofluorescence analyses on cells, 15,000 B16F10 cells were seeded on 12 mm glass coverslips in 24-well plates. 24 h after seeding, cells were treated for 24 h with 0.5 µM mitoTRAM-34, 5 µM rev-mitoTRAM or the corresponding amount of DMSO (0.1%). For rescue experiments, cells were co-treated with 2.5 µg/ml Rho/Rac/Cdc42 Activator I (Cytoskeleton, Inc. #CN-04A). After treatment, cells were washed 1x in PBS, fixed in 3.8% PFA for 15 min, washed 3x in PBS, permeabilized for 10 min in 0.1% Triton X-100 (Sigma-Aldrich) in PBS, washed 3x in PBS, blocked for 1 h in 1% BSA + 5% goat serum in PBS and incubated overnight at 4 °C in 1% BSA + 5% goat serum in PBS with anti-TOM-20 (1:500, SCBT #sc-11415). The next day, cells were washed 3x in PBS and incubated for 1 h at room temperature with secondary antibody anti-rabbit AF488, ThermoFisher #A-11008, 1:1000 in 5% goat serum in PBS) or with 150 nM AF488 Phalloidin (ThermoFisher #A12379) in 5% goat serum in PBS. Samples were washed 3x in PBS and mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) and images were acquired with a Zeiss LSM900 Airyscan2 confocal microscope.