β2 spectrin

Inner ears were fixed for 2 hours in a solution of 4% paraformaldehyde prepared in 67mM Sorenson’s phosphate buffer (pH 7.4). For immunofluorescent labeling, inner ear sensory organs were micro-dissected to expose the sensory epithelia and permeabilized and blocked using blocking solution (5% donkey serum, 1% BSA, and PBS) supplemented with Triton X-100 to 0.5%. Primary antibodies and phalloidin Alexa Fluor 488 (Invitrogen A12379) were diluted in blocking solution supplemented with Tween-20 to 0.1% and incubated with the tissue overnight at 4°C. Tissue was washed thoroughly with PBS-T (PBS and 0.05% Tween-20), followed by incubation with species-specific, Alexa Fluor-conjugated (Invitrogen) or DyLight-conjugated (Jackson ImmunoResearch) secondary antibodies. Tissue was subsequently washed with PBS-T, mounted using Prolong Gold (Molecular Probes, P36930), and imaged via structured illumination microscopy using a Zeiss Axio Imager M.2 with ApoTome.2 attachment. Images were collected with Zeiss Zen software, and figures were prepared with Adobe Illustrator. The following commercial antibodies were used in this study: β2-Spectrin (BD Biosciences 612562); Oncomodulin (Santa Cruz Sc7446). The Celsr1 antibody was provided by D. Devenport and has been previously described (Devenport and Fuchs, 2008 (link)).