Ab150073

Cochleae were fixed overnight at 4 °C in 4% paraformaldehyde (PFA) (Santa Cruz Biotechnology, Dallas, TX, sc-281692). Adult tissue was decalcified in 0.2 M EDTA (pH 7.3) for 14 days at 4 °C. For whole-mount antigen retrieval, dissected cochleae were immersed in 30% sucrose, flash frozen in liquid nitrogen, allowed to thaw, washed in PBS, and blocked for one hour in 1% Triton X-100/5% horse serum (Sigma) in PBS, and incubated in primary antibodies overnight. Refer to Table S1 for information regarding primary antibodies used in this study. Tissue was subsequently washed in PBS and incubated for two hours at room temperature in Alexa Fluor 647 Phalloidin (Invitrogen, A22287), [1:100]) Alexa Fluor secondary antibodies (Invitrogen, ab150073, ab 11056, ab21202, and ab150074, [1:1000]), counterstained with 4‘,6-diamidino-2-phenylindole (DAPI), washed again in PBS and mounted in Fluorogel in Tris buffer (Electron Microscopy Sciences).

After treatments, the tissues were transferred to Tissue-Tek optimal cutting temperature (O.C.T, Sakura Finetek, Tokyo, Japan) compound to snap freeze for immunohistology. IHC was done as previously described [32 (link),34 (link)]. Briefly, 10 µm tissue sections were air-dried on the slide glasses (Matsunami Glass, Osaka, Japan) for 40 min at RT, then washed with tris-buffered saline with 0.1% Tween20 (1× TBST) for 10 min. Tissues were blocked with 3% bovine serum albumin (BSA Gemini, Bio-Products, West Sacramento, CA, U.S.A.)/1× TBST for 1 h followed by overnight incubation with vimentin diluted at 1:300 (Abcam, Cat. No.: ab92547, Lot No.: GR3186827-13) and cytokeratin-18 diluted at 1:800 (Abcam, Cat. No: ab668, Lot No: GR3196069-6) in 3% BSA/1× TBST at 4°C. The next day, tissues were washed 3 times with 1× TBST for 10 min each, then treated with secondary antibodies diluted at 1:1000 (Alexa Fluor® 488, Abcam, Cat. No.: ab150073, Lot: GR269274-4 and Alexa Fluor™ 594, Invitrogen, Cat. No.: A11005, Lot: 2179228, respectively) in 3% BSA/1× TBST for 3 h at RT. Then, tissues were stained with NucBlue™ Fixed Cell ReadyProbes™ (DAPI, Invitrogen, Cat. No: R37606, Lot No: 2216969) for 5 min, and washed 3 times with 1× TBST for 10 min each. The images were taken with a Keyence microscope (Keyence Corp., Osaka, Japan).