Cacodylate buffer

After euthanasia both auditory bullae from each animal were isolated and opened to expose the cochlea. Fixative (2.5% Glutaraldehyde in 0.01 M Cacodylate buffer) (Agar Scientific, UK) was injected gently into the cochlea via the round window, exiting via a hole made in the bone at the apex. Time between euthanasia and cochlea fixation was <5 min in all cases. The bullae were then immersed in fixative and fixation continued for 2 hours at room temperature. They were then decalcified in a solution of 4% EDTA in Cacodylate buffer for 48 h at 4°C. The IHCs of several control animals were examined to check that fixation had not caused artefactual swelling of the nerve terminals. Tissue from noise-damaged animals was prepared in parallel with this control tissue.

For SEM analysis cells cultured on the NCD or glass substrate were fixed, dehydrated, within the culturing dish as previously described (Spira et al., 2003 (link)). Briefly, hippocampal primary cultured cells were fixed by 3% glutaraldehyde (Electron Microscopy Science) in cacodylate buffer (Agar Scientific, Stansted) at pH7.4. The cells were then washed in cold 0.1 M cacodylate buffer, pH7.4. The cells were post-fixed in 1% osmium tetroxide (Electron Microscopy Science) and 1.5% K₃Fe(CN)₆ (Sigma–Aldrich). Dehydration was carried out through a series of ethanol solutions and washed two times for 30 min with fresh 100% EtOH before critical point drying with liquid CO₂ in a SAMDRI-PVT-3D (Tousimis, USA). Once dry the samples were sputtered with gold in an SPI-Module^TM Sputter Coater Module (SPI Supplies, USA). Images were taken with an Extra High Resolution Scanning Electron Microscopy MagellanTM 400L using an accelerating voltage of 5 kV.