Immunohistochemistry of Vestibular Efferents

Mice were anesthetized with IP ketamine/xylazine (100/10 mg/kg) and perfused with 4% (w/v) PFA freshly prepared in phosphate-buffered saline (PBS, 0.9% NaCl in 0.1 M PB). As described previously^{11 (link)}, vestibular labyrinths were extracted from the temporal bone of three C57BL/6 mice and immunohistochemistry was performed on microdissected vestibular organs with antibodies to ChAT (1:100, Millipore) and calretinin (1:1000; Millipore). Anti-ChAT was used to label vestibular efferent fibres and varicosities while anti-calretinin was used primarily to counterstain hair cells in the otolithic macula and calyx afferents in the crista central zone^{11 (link)}. Whole mount projections were acquired either with a Zeiss Axio Imager motorized upright multifluorescent microscope fitted with an Apotome slider system (Zeiss Imaging Systems, Oberkochen, Germany) or Olympus FV1000 Laser Scanning Confocal microscope. Images were postprocessed using Axiovision, Olympus FV1000, and/or Adobe Photoshop CS6 (Adobe Systems, San Jose, CA) software to adjust contrast and brightness within the linear range.