High-resolution immunofluorescence imaging of mouse cochlear hair bundles

High-resolution immunofluorescence imaging of cochlear hair bundles faces many challenges due to the hair bundle’s small dimensions, fragile nature, and complex organization. Here, we describe an optimized protocol for hair-bundle protein immunostaining and localization. We detail the steps and solutions for extracting and fixing the mouse inner ear and for dissecting the organ of Corti. We further emphasize the optimal permeabilization, blocking, staining, and mounting conditions as well as the parameters for high-resolution microscopy imaging. For complete details on the use and execution of this protocol, please refer to Trouillet et al. (2021). [Display omitted] •Techniques for dissecting the mouse cochlea and the organ of Corti•Dissection, permeabilization, blocking parameters to detect hair bundle proteins•Mounting method to localize protein in the hair bundles Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. High-resolution immunofluorescence imaging of cochlear hair bundles can be difficult because of the hair bundle’s small dimensions, fragile nature, and complex organization. Here, we describe an optimized protocol for hair-bundle protein immunostaining and localization. We detail the steps and solutions for extracting and fixation of the mouse inner ear and the dissection of the organ of Corti. We note the optimal permeabilization, blocking, staining, and mounting conditions and the parameters for high-resolution microscopy imaging.