Abstract
Fluorescence recovery after photobleaching (FRAP) has been widely used to monitor membrane properties by measuring the lateral diffusion of fluorescent particles. This protocol describes how to perform two-photon FRAP on the stereocilia of live cochlear inner hair cells using a lipophilic dye, di-3-ANEPPDHQ, to assess the stereociliary membrane diffusivity. We also detail two-photon FRAP microscope setup and calibration, as well as FRAP parameter setting and data analysis. For complete details on the use and execution of this protocol, please refer to George et al. (2020).