Immunohistochemical Analysis of Prestin Expression in Mouse Cochlea

Mice were cardiac perfused with 4% paraformaldehyde and cochleae extracted. After post-fixation and decalcification, cochleae were dissected following the Eaton-Peabody Laboratory cochlear dissection protocol [28 (link)]. In order to detect prestin, N-terminal prestin rabbit antisera [58 (link)] was used at 1:1000 with goat anti-rabbit Alexa Fluor 488 (Thermo) as the secondary antibody at 1:500. Alexa 546-conjugated phalloidin and Hoechst 33342 (Thermo) were also used to stain actin and nuclei, respectively, as described before [45 (link)]. Stained cochlear sections were mounted onto slides using Dako fluorescent mounting medium (Agilent). Images were captured on a Nikon A1R confocal microscope with Plan Fluor 10X and Plan Apo 20X objectives (Nikon) controlled by NIS Element software. Basilar membrane length was measured using ImageJ, and the numbers of remaining OHCs determined. A mouse cochlear place-frequency map [32 (link)] was used to determine the corresponding frequencies.