Sc 365949

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.

NRF2 nuclear translocation was examined by immunostaining as described previously [24 (link)]. In short, cells were fixed in 3.7% PFA/PBS for 10 minutes at room temperature and were permeabilized using 100 μl of 0.5% Triton X100/PBS for 10 minutes and blocked using 200μl of blocking buffer (2% bovine serum albumin (BSA), 0.2% Triton X100, PBS) for one hour at room temperature. A 100 μl of NRF2 antibody (sc-365949; Santa Cruz Biotechnology, TX, USA) in blocking buffer (1:100 dilution) was subsequently applied and incubated for four hours at room temperature and 4°C overnight. The next day, a 100 μl of anti-mouse secondary antibody, Alexa Fluor 647 (A21235; Invitrogen^™, CA, USA) in blocking buffer (1:250 dilution) was applied and incubated for one hour at 37°C in a humidified incubator. Cells were then washed three times with PBS for three minutes and a 100 μl of Hoechst 33342 nuclei stain in PBS (1:10,000 dilution) was applied for 10 minutes. The transwell membrane containing the cells was excised from the insert using No.11 scalpel blade in a clockwise motion and mounted directly on a glass slide. 20 μl of ProLong^™ Gold Antifade Mountant (Invitrogen^™, CA, USA) was applied, and coverslip was placed gently on top. Images were visualized and captured on the Zeiss Confocal LSM 710 and the mean intensity of nuclear NRF2 was quantified by the Image J software.