Anti myosin viia

After the measurement of hearing, mice were sacrificed and dislodged the encapsulated cochlea carefully. Dissected inner ears were fixed with 4% paraformaldehyde, decalcified in EDTA decalcifying solution, microdissected the cochlear epithelium, and then flatten the specimen to orient the sensory hair cell surface side up. Sections were blocked for 1 h with 10% goat serum. The primary antibody, anti-Myosin VIIa (1:50, Abcam) and 4-HNE (1:25, Abcam), anti-GLUT1 (1:500, Abcam), anti-LDHA (1:250, Proteintech), or anti-HIF-1α (1:100 Proteintech), were incubated overnight. Next day, sections were incubated with secondary antibodies for 1 h at room temperature: CoraLite594-conjugated goat anti-rabbit IgG(H+L) (1:250, Proteintech), CoraLite488-conjugated goat anti-mouse IgG(H+L) (1:250, Proteintech). Sections were sealed with mounting medium containing DAPI (Abcam, United States), and photographs were taken by a confocal microscopy (Leica, Italy).

The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Nonspecific binding sites were blocked, and an additional permeabilisation step was performed for 1 h in 0.2% Triton X-100 and 1% bovine serum albumin. The cells were incubated overnight with a primary antibody at 4 °C. Following this incubation, the fluorescein isothiocyanate- and tetramethylrhodamine-conjugated species secondary antibodies (1:500, Invitrogen, Carlsbad, CA, USA) were used to detect the primary antibodies. The primary antibodies used in this study were anti-MyosinVIIa (1:500, Abcam, Cambridge, MA, USA) and anti-Espin (1:500, Abcam). Nuclei were visualised using the Hoechst stain (Invitrogen). For FM1-43 staining (Invitrogen), the cells were immersed in the stain solution on ice for 1 min according to the manufacturer’s instructions. We used FM 1-43 staining to demonstrate that the iPSC-derived differentiated HC-like cells exhibit the expression of not only HC markers (MYO7A and ESPN) but also specific ion channels in HCs⁵⁷ . It was reported that FM 1-43 can be used to characterise HCs^{55 (link)} and examine the recycling of synaptic vesicle-associated activities⁵⁶ .