Alexa fluor 568 conjugated phalloidin

The animals were anesthetized with a mixture of ketamine (100 mg/kg, i.m.) and xylazine (15 mg/kg, i.m.) and sacrificed by cervical dislocation. Cochleae were quickly removed and fixed in 4% paraformaldehyde in PBS at 4 °C for 2 h. Then, the basilar membrane, the cochlear lateral wall, and the spiral ganglion neurons were microdissected out. The specimens were immersed overnight at 4 °C in a polyclonal primary rabbit anti-Cox-2 antibody (abcam, USA) diluted in 1% Triton X-100 and 5% donkey serum in 0.1 M PBS (1:100). After three washes in PBS, specimens were incubated in a secondary donkey anti-rabbit antibody conjugated with Alexa Fluor 488 diluted in PBS (1:200) for 2 h at room temperature. Then, specimens were incubated for 1 h at room temperature with Alexa Fluor 568-conjugated phalloidin (Sigma, USA) to stain the hair cell stereocilia and cuticular plate. After rinsing in PBS, the specimens were immersed for 10 min in fresh DAPI solution to label the nuclei of cells in the specimen. To mark spiral ganglion neurons, the specimens were co-incubated with a monoclonal primary mouse anti-neurofilament antibody (Sigma, USA) and a secondary donkey anti-mouse antibody conjugated with TRITC. The specimens were subsequently coverslipped with a SlowFade® anti-fading mounting solution and examined under a confocal microscope (Carl Zeiss, Germany).

Cells were grown on 35-mm-diameter cell culture dishes (FluoroDish, World Precision Instruments, Inc) coated with poly-L-lysine (Sigma, 5 μg cm⁻²) and subjected to blebbing solutions identical to those used for electrophysiological study. Cells were then fixed for 15 min using 4% paraformaldehyde at room temperature. Subsequently, control cells or those expressing PIEZO1 or MscL GFP fusion proteins, were permeabilized using 0.5% Triton X-100 (Sigma) for 2 min and stained for 20 min at room temperature with Alexa Fluor 568 conjugated phalloidin (Sigma). Confocal images were made using an inverted confocal microscope (LSM 700; Carl Zeiss) equipped with a water immersion long working distance objective (× 63; NA 1.15; Carl Zeiss). Both 488 and 555 nm lasers were used to excite the respective fluorophores (GFP, Alexa Fluor 568).