Tom20 fl 145

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.