Alexa fluor 488 anti rabbit

Cochlear samples were prepared using a procedure similar to that used for the histological hair cell analysis. The decalcified samples of cochlea were rinsed in 10 mM PBS containing 10% and 30% sucrose, embedded in Tissue-Tek O.T.C. Compound (Sakura Finetechnical), and frozen in liquid nitrogen. Thin sections (8 μm) were obtained with a cryostat (CM3000, Leica Instruments). Immunohistochemistry was performed according to a previously described procedure with modification42 (link). Briefly, tissue sections were rinsed in 0.1% Triton X-100/Tris buffered saline (TBS), blocked with 3% bovine serum albumin/0.3% Triton X-100/TBS (TBST) for 30 min at room temperature, and incubated with an unconjugated AffiniPure Fab fragment anti-mouse IgG (1:10, Jackson ImmunoResearch) for 2 hr at room temperature. The sections were incubated overnight with primary antibodies (anti-4HNE; 1:400, JaICA. anti-Myosin 7a; 1:500, Abcam) diluted in 0.3% TBST at 4 °C. All of the sections were washed three times in 0.1% TBST and incubated with secondary antibodies (Cy3 anti-mouse; 1:500, Jackson ImmunoResearch, Alexa Fluor 488 anti-rabbit; 1:500, Jackson ImmunoResearch) for 1 hr at room temperature under light-protected conditions. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:2,000).

Directly after cell fixation using 4% PFA, cells were treated with 0.05% Triton X-100 (Sigma–Aldrich, T8787) for 3 min at RT to increase membrane permeability and were blocked for non-specific binding using 2% BSA for 1 h at RT. For F-actin staining, NHBE cells were incubated with Alexa Fluor 568 Phalloidin (ThermoFisher Scientific, A12380) diluted with PBS (ratio of 1:200) for 40 min at RT. For DAPI nucleic acid staining cells were incubated with DAPI solution (ThermoFisher Scientific, D1306), diluted with PBS (ratio of 1:400) for 5 min at RT. For tight junction proteins, Zonula occludens-1 (ZO1), cells were incubated with the primary antibodies rabbit anti-ZO1 (ThermoFisher Scientific, 617300) diluted with PBS (ratio of 1:200) overnight at 4°C, followed by incubation with secondary antibody Alexa Fluor 488 anti-rabbit (Jackson ImmunoResearch, 111-545-144), diluted with PBS (ratio of 1:400) for 1 h at RT. After each step, cells were washed three times with PBS. Finally, confocal microscopy imaging of fluorescent immunostaining was performed (Nikon Eclipse Ti with spinning disk, Yokogawa, Japan).

For LC3 staining, samples were fixed in ice‐cold methanol for 5 min at room temperature, followed by rehydration in PBS and processing for immunofluorescence. Serum starvation in 0.5% FBS was included as a control for induction of LC3 puncta. Fixed samples were rinsed 3–5 times in blocking buffer (2% BSA, 0.1% Triton X‐100, 0.1% NaN₃ in PBS). Samples were incubated with LC3 diluted in blocking buffer for 1 h at room temperature. Samples were rinsed 3–5 times in blocking buffer for 5–10 min each before incubating in secondary antibodies (1 : 500) for 45 min at room temperature. Secondary antibodies and phalloidin were diluted in blocking buffer. Samples were rinsed 3–5 times in PBS, 5–10 min each, and then mounted in Mowiol mounting medium (0.1 M Tris/HCl, pH 8.5, 25% glycerol, 10% Mowiol 4‐88; 475904, Calbiochem, 2% DABCO; D2522, Sigma). All secondary fluorescently labelled antibodies were highly cross‐absorbed secondary antibodies from Jackson ImmunoResearch, Alexa Fluor^® 488 Anti‐Rabbit (711‐545‐152) and Rhodamine (TRITC) Anti‐Mouse (715‐025‐151), used at 1 : 500.