Tcs sp5ii laser scanning confocal micro scope

Cell-permeable Ca²⁺ indicator Fluo-4 AM (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) were used to monitor levels of intracellular Ca²⁺, based on the literature (36 (link)). Following immersion in HBSS without Ca²⁺ and Mg²⁺ for 20 min at 20-24°C, the organ explants were immersed in dye (cat. no. F14201; Invitrogen; Thermo Fisher Scientific, Inc.) for 25 min following Fluo4-AM loading at 24°C. Subsequently, the explants were fixed in 4% paraformaldehyde for 30 min at 20-24°C. Phalloidin (1:200) was applied for 1 h at 20-24°C and protected from light. For measurement of Fluo-4 Ca²⁺ signals, organs were imaged under a BX41 microscope at ×400 magnification (Olympus Corporation) with epifluorescence, and the images were obtained using a TCS-SP5II laser-scanning confocal micro-scope (×400 magnification; Leica Biosystems GmbH) in 20 consecutive fields.

After the ABR test, the temporal bones were harvested. Each bulla was opened using rongeurs to expose the cochlea. The oval and round windows were then opened. Following the creation of a hole in the cochlea apex, 4% paraformaldehyde was perfused through the cochlea for at least 24 h. The cochlea was decalcified in 4% EDTA for 7 days at 4°C. Subsequently, the basilar membrane was dissected under a dissecting microscope, and the stria vascularis and tectorial membrane were removed. To identify F-actin in the organ of Corti, tetramethyl rhodamine isothiocyanate (TRITC) (Sigma-Aldrich) was applied for 20 min at room temperature and protected from light. The specimens were then rinsed 3 times with 0.01 M phosphate-buffered saline (PBS) (pH 7.4). Fluorescence signals from the hair cells were counted under a BX41 microscope with epifluorescence (Olympus, Tokyo, Japan), and the images were obtained with TCS-SP5II laser-scanning confocal microscope (Leica Biosystems, Wetzlar, Germany). Three rows of the outer hair cells (OHCs) were counted from the apex through the basilar turn of the cochlea under ×200 magnification in 20 consecutive fields.