Tcs sp5 sted microscope

For microscopy, embryos were anesthetized with Tricaine (Sigma) and fixed using 0.5% low-melting-point agarose in glass-bottomed dishes (MatTek). Images were taken by using a Leica TCS SP5-STED microscope. Image stacks were taken with a 10× objective and 1.3 digital zoom. For time-lapse imaging, embryos (kept in the chorion) were fixed in 0.5% low-melting-point agarose, and a heated-stage chamber was used to keep embryos at 28.5°C. Image stacks were taken every 25 minutes. Images were processed using Leica software for automatic stitching and ImageJ to create overlays and maximum projections.

Imaging of the tissue sections was carried out on a Leica TCS SP5 STED microscope (used in confocal mode) using either a 40×/1.1 or a 63×/1.2 water immersion objective. Sytox Orange was excited by using a 561 laser. A HyD detector (nm range) was employed to capture the signals.

Rat primary hippocampal neuron cultures (Fig 4 and Appendix Figs S12–S14) were prepared as described before (Opazo et al, 2010; Beaudoin et al, 2012) and were cultured either under standard conditions, or in Banker arrangements, locally separated from the astrocyte feeder layer (as described in Kaech & Banker, 2006). The neurons, plated on poly‐L‐lysine coated cover slips, were fixed in PFA (pH 7, pH 4/5 or with Et‐OH) or glyoxal for 60 min and were subsequently quenched for 30 min in 100 mM NH₄Cl. The pH of the glyoxal solution used for fixation is presented in Table 1. For each antibody, we used the pH that provided a brighter immunostaining. Permeabilization and background epitope blocking were achieved by incubating the neurons for 15 min in blocking solution, containing 2.5% BSA and 0.1% Triton X‐100 in PBS. The samples were incubated with primary antibodies diluted in blocking solution, for 60 min at room temperature. Table 2 presents the antibodies and their dilutions from 1 mg/ml stocks. After washing another 15 min in blocking solution, secondary antibodies were applied for 60 min, at room temperature. Subsequent washing in high‐salt PBS (500 mM NaCl) and PBS was followed by embedding in Mowiol. The samples were imaged with a STED TCS SP5 microscope (Leica).

Organs of Corti (Appendix Fig S17) were dissected from P14 to P18 wild‐type mice in ice‐cold HBSS (5.36 mM KCl, 141.7 mM NaCl, 10 mM HEPES, 34 mM l‐glutamine, 6.9 mM d‐glucose, 1 mM MgCl₂, 0.5 mM MgSO₄, pH 7.4). The inner hair cells were stimulated by incubating the tissue for 3 min in HBSS with high potassium (65.36 mM KCl) at 37°C. Afterward, the organs were fixed for 30 min on ice and for another 30 min at room temperature. The subsequent quenching was performed for 30 min in 100 mM NH₄Cl and 100 mM glycine. The organs were then permeabilized and blocked for 30 min with PBS containing 0.5% Triton X‐100 and 2.5% BSA. The primary antibodies mouse anti‐otoferlin (Abcam #ab53233), diluted 1:350, and rabbit anti‐ribeye (Synaptic Systems #192003), diluted 1:1,500, were applied for 60 min. After 30 min of washing, the organs were incubated in secondary antibodies for 60 min. Atto647‐labeled goat anti‐mouse (1:250, Sigma‐Aldrich #50185) and the Cy2‐labeled goat anti‐rabbit (1:100, Dianova #111‐225‐144) secondary antibodies were used. Washing in high‐salt PBS and PBS was followed by embedding in melamine, as described previously (Revelo et al, 2014). Organs were then cut into 200‐nm thin sections using a Leica EM UC6 ultramicrotome. The sections were embedded in Mowiol and were imaged using a STED TCS SP5 microscope (Leica).