Pm045

Immunocytochemistry was used to probe the cellular localization of Opa1, Cytochrome c oxidase (Cox) and some autophagic and antioxidant markers in cryostat sections using antibodies recognizing Opa1 (1/125; Abcam #ab42364 RRID:AB_944549), Cytochrome c oxidase subunit I (1/500; Invitrogen, #459600 RRID:AB-1501840), p62 (1/1000, MBL International #PM045 RRID:AB_1279301) and Nrf2 (1/1000, Santa Cruz Biotechnology # sc-365949 RRID:AB_10917561). Anti-parvalbumin (1/500; Swant, Bellinzona, Switzerland, #PV235 RRID:AB_10000343) was used to label the hair cells and the spiral ganglion neurons. All secondary antibodies were used at a dilution of 1/1000. This included donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 or Alexa 568 (Molecular Probes #A-21202 RRID:AB-141607, #A-21206 RRID:AB-2535792, #A-10037 RRID:AB-2534013). DNA was stained by Hoechst 33342 (0.002% wt:vol, Sigma, Saint Louis, Missouri, USA). Fluorescent tags were visualized using a confocal microscope (ZEISS LSM 880 Airyscan). In control specimens without primary antibodies, neither Alexa 488 nor 568 fluorescent tags were observed. Immunocytochemistry analysis required 4 to 5 cochleae per age and strain (Table S1). All experiments were performed in triplicate.

Paraformaldehyde-fixed NRK, HEK293, and HEK293T cells were visualized using a Leica TCS SP8 DIVE confocal microscope (Leica) equipped with an Airyscan detector and a 63× oil immersion objective (1.4 numerical aperture; Leica). Images were acquired in Airyscan mode, and processed with LAS X software (Leica). As indicated, samples were probed with anti-p62 (MBL, PM045; dilution 1:500), anti-HA (Santa Cruz, F-7; dilution 1:100), anti-FK2 (MBL, D058-3; dilution 1:500) or anti-LC3A/B antibody (CST, 12741S; dilution 1:100). Imaging conditions and intensity scales were similar for each group presented together.