Mowiol dabco

For immunostaining cells were seeded on cover slips and after treatment fixed with 4% PFA. The fixed cells were washed with PBS and then permeabilized and blocked with a buffer containing 10% goat serum and 0.2% Triton X-100 in PBS. Cells were afterwards incubated with the primary antibody in antibody-dilution-buffer (3% goat serum and 0.05% Tween20 in PBS) for 1 h at RT. After wash and 10 minutes incubation in the blocking buffer, samples were incubated with the corresponding secondary antibody diluted in antibody-dilution-buffer for another 50–60 min. In the final step, the cover slips were mounted onto glass-slides using 2.5% Mowiol-DABCO (Carl Roth, Karlsruhe, Germany). Images were acquired on a TCS SP5 confocal microscope (Leica Biosystems, Wetzlar, Germany) using a 63x oil-immersion UV objective with a numerical aperture of 1.4 or, for superresolution imaging, on a Zeiss (Oberkochen, Germany) ELYRA S.1 SR-SIM structured illumination platform using a Plan-Apochromat 63x oil-immersion objective with a numerical aperture of 1.4. Reconstruction of superresolution images were performed using the ZEN image-processing platform with a SIM module.

Mitochondrial morphology in MEF cells and patient fibroblasts was visualized using MitoTracker^TM Red CMXRos (Invitrogen, United States). The cells were seeded onto sterile cover slips the day prior to the experiment. Cells were incubated in 200 nM MitoTracker^TM Red CMXRos diluted in fresh DMEM for 30 min at 37°C. For SO₃^2– treatments, the respective SO₃^2– concentration was added to the medium 30 min prior to the MitoTracker and incubated at 37°C. Afterward, the cells were washed carefully with PBS before fixation in 4% PFA for 15 min at 4°C. Remaining PFA was removed by three washing steps with PBS (3 × 5 min). Finally, cover slips were mounted on slides with Mowiol/DABCO (Carl Roth). Images were acquired using a Nikon A1 confocal laser scanning microscope. Images were processed using ImageJ software.