Alexa 546 conjugated phalloidin

Cochlear whole mounts were prepared as above, except 4% paraformaldehyde was used for the cardiac perfusion and post fixation. In order to detect both WT and ∆IDR prestin, N-terminal prestin antisera (anti-N-mprestin)^{10 (link)}, or C-terminal prestin antibody (anti-C-mprestin)^{10 (link)}, was used at 1:1000, followed by goat anti-rabbit Alexa 488 secondary antibody at 1:500 (ThermoFisher; AB 2576217). Alexa 546-conjugated phalloidin (ThermoFisher; AB 2632953) and Hoechst 33342 (ThermoFisher) were also used to stain actin and nuclei, respectively. Images were captured on a Nikon A1R confocal microscope with a Plan Apo 20X objective or Plan Apo 60X oil objective (Nikon) controlled by NIS Element software (Nikon). Additional details are provided in a previous publication^{2 (link)}.

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.

The following immunofluorescence procedure was performed to evaluate the zyxin and F-actin distribution. Primary HGFs (1000 cells) were grown on coverslips for 24 h at 37 °C and then incubated with retraction agents diluted in the culture medium (1 mg/mL) for 24 h at 37 °C. Next, the fibroblasts were washed with PBS (BioShop, Mainway, Burlington, Canada) for 5 min at room temperature, fixed using 4% PFA (Sigma-Aldrich) in PBS (10 min at room temperature), permeabilized with 1% triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS (v/v) (3 × 3 min at room temperature) and blocked with 1% FBS in PBS (1 h at 37 °C). All washing steps were performed with PBS. After overnight incubation with primary mouse monoclonal antizyxin antibody (diluted 1:200) (Abcam, Cambridge, UK) at 4 °C, the cells were washed with PBS (2 × 10 min at room temperature) and labelled with the secondary antibody—Fluorescein (FITC)-conjugated AffiniPure Fragment Donkey Anti-Mouse IgG (for 60 min at room temperature; diluted 1:100) (Jackson ImmunoResearch, Cambridgeshire, UK) mixed with Alexa 546-conjugated phalloidin (at a concentration of 2 μg/mL) (Life Technologies, Carlsbad, CA, USA). Then, the cells were mounted in a fluorescence mounting medium (DAKO). An Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokio, Japan) with 60x magnification was used for imaging.