Deconvolution microscope

Cochlear explants were dissected from P3 mice and cultured in DMEM/F12 medium (Cat.# 21041025, Life Technologies Corporation) overnight at 37°C in a 5% CO2 humidified atmosphere. Then, the samples were incubated in DMEM/F12 medium containing various concentrations of cisplatin (Cat.# 232120, Millipore Sigma, dissolved in DMEM/F12) for 2 days at 37°C. Cimetidine (Cat.# AAJ6282506, Fisher Scientific) was also dissolved in DMEM/F12 and added to the cochlear explants prior to adding of cisplatin. To monitor the cisplatin uptake in hair cells, the cochlear explants were incubated in DMEM/F12 medium containing 2 μg/mL of TR–cisplatin (Ursa BioScience) for various amounts of time. Stacked images were then captured using a deconvolution microscope (Leica) with a 63 × objective (HCX APO L63X/0.90 Water). The images were then deconvoluted using the blind deconvolution method.

Immunostaining was performed as described previously (Liu et al., 2018 (link)). In brief, the cochlear explants were fixed in 4% PFA for 20 min at room temperature and then washed three times for 5 min each in HBSS. The tectorial membrane was then removed and the samples were blocked in blocking buffer (5% bovine serum albumin and 0.5% Triton X-100 in HBSS solution) for 20 min at room temperature. Then, the samples were incubated with primary antibodies diluted in antibody dilution buffer (1% BSA and 0.1% Triton X-100 in HBSS solution) overnight at 4°C. After being washed with HBSS, the samples were incubated with secondary antibodies for 2 h at room temperature. Then, the samples were mounted in ProLong ^® Antifade Reagents (Cat.# P36971, Life technologies Corporation). Stacked images were then captured using a deconvolution microscope (Leica) with a 20 × objective (HC PL FLUOTAR 20x/0.55) or a 100 × objective (HCX PL APO 100x/1.40-0.70 OIL). Antibodies used in this study were anti-β2 spectrin (1:200, Cat.# sc-136074, Santa Cruz) and Alexa Fluor 488 goat anti-mouse (1:2,000, Cat.# A11017, Life technologies Corporation).

Cells were adhered onto coverslips and incubated with Remodelin at 1 μM for 2 days. To visualize nuclei, cells were subsequently fixed with 4% paraformaldehyde in PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI). Images of cells were acquired using a Deconvolution microscope (Leica). CellProfiler software was used to quantify nuclear circularity and nuclear area from DAPI staining pictures, using the ‘object size shape’ measurement.