63 1.4n a oil immersion objective

We have previously developed and extensively tested a model enabling the precise determination of the mPTP sensitivity to oxidant stress in intact cardiac myocytes [18 (link),19 (link),25 (link)]. Briefly, small numbers of mitochondria inside isolated cardiomyocytes were exposed in situ to conditions of oxidative stress by repetitive (2 Hz) laser line-scanning (with imaging) of a single row of mitochondria in a cell loaded with 100 nM tetramethylrhodamine methyl ester (TMRM), using a Zeiss LSM 510 inverted confocal microscope (Carl Zeiss Inc., Jena, Germany) with excitation at 543 nm and collecting emission at >560 nm using a Zeiss 63×/1.4N.A.oil immersion objective. This results in incremental, additive exposure of only the laser exposed area to the photodynamic production of ROS and consequent mPTP induction. The ROS-threshold for mPTP induction (t_mPTP) was measured as the average time necessary to induce the mPTP due to the local buildup of ROS in a targeted row consisting of ~25 mitochondria. MetaMorph image analysis software (Molecular Devices Corp., Downingtown, PA, USA) was used to calculate t_mPTP. The data are mean ± SEM.

For immunostaining, primary islets were plated on poly‐D‐lysine–coated coverslips 72 h prior to imaging. Cells were fixed at 4% vol/vol paraformaldehyde (PFA) for 15 min at room temperature. After washing two times in PBS, cells were incubated in 50 mM NH₄Cl for 10 min followed by a 10 min 20 mM glycine incubation. Cells were permeabilized (2 μl/ml Triton X‐100 and 0.5 mg/ml sodium deoxycholate in PBS, pH 7.4) for 15 min at room temperature. Subsequently, cells were blocked with 10% FBS for 1 h at room temperature. Cells were incubated with 1:200 primary antibody of GPR75/Mortalin (Abcam, ab110325) at 4°C overnight. The next day, cells were washed in PBS and incubated with 1:200 primary antibody of TOM20 (Santa Cruz, Tom20 FL‐145) at 4°C for overnight. On the last day, cells were incubated with 1:500 Anti‐Rabbit Alexa Fluor 488 (Thermo, A11008) or Anti‐Mouse Alexa Fluor 568 antibodies (Thermo, A11004) for 1 h at room temperature, and samples were kept in PBS. A Zeiss LSM 880 confocal microscope in Airyscan mode was used for super‐resolution imaging, with a 488 nm Argon laser and Zeiss 63×/1.4NA oil immersion objective.