Immunofluorescence Imaging of Mouse Cochlea

Cochleae from adult mice were prepared and examined as described previously (85 (link), 86 (link)). All images were acquired with a confocal microscope (LSM 700 or 710, Zeiss). Samples were stained with phalloidin (A12379 or A12380, Invitrogen) or with antibodies for other markers. The following primary antibodies were used: anti–myosin VI (1:400; 25-6791; Proteus Bioscience), anti–myosin VIIa (5 μg/μl;138-1-s, Developmental Studies Hybridoma Bank), anti-Tuj1 (1:250; 801201, BioLegend), anti-pERK (1:400; 9101S, Cell Signaling Technology), or anti-Ctbp2 (1:500; 612044, BD Transduction). Anti-rabbit Alexa Fluor 488 (1:400; A11034) and anti-mouse Alexa Fluor 647 (1:400; A31571) secondary antibodies were purchased from Invitrogen. ProLong gold antifade reagent with 4′,6-diamidino-2-phenylindole (P36941, Invitrogen) was used to counterstain nuclei.