1200ex microscope

Organ of Corti explants were dissected at P4 in L-15 medium and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2), supplemented with 2 mM CaCl₂. The fixative was supplemented with 1% tannic acid to visualize links (1–2 h at room temperature) or 5% tannic acid for stereocilia surface coat observation (applied overnight at 4 °C). Following a triple rinse in cacodylate buffer, they were postfixed with 1% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 M cacodylate buffer for 2 h at room temperature in the dark. Explants were then washed three times in 0.1 Organ of Corti explants were dissected cacodylate buffer (pH 7.2), then briefly washed in distilled water, dehydrated in an ascending series of ethanol, equilibrated in propylene oxide and infiltrated and embedded in epoxy resin (Araldite 502/Embed-812 embedding media). Tissue pieces were positioned in molds and polymerized in the oven at 60 °C for 48 h.
The resin blocks were sectioned at 60–80 nm steps using a Reichert Ultracut S ultramicrotome. The sections were mounted on copper Formvar/Carbon-coated grids, stained with 2% uranyl acetate followed by lead citrate (Reynolds, 1963), and viewed with a JEOL 1200EX microscope operating at 80 kV. Images were captured with an Advanced Microscopy Techniques camera system at 3488 × 2580 pixel resolution.

Samples were stained and visualized essentially as described previously [10 (link), 90 (link)]. Aliquots of freshly thawed protein samples were adsorbed (10 μl) neat onto formvar-coated copper grids (Electron Microscopy Sciences, Hatfield, PA) for 1 min before 10 μl of 0.25% glutaraldehyde (ThermoFisher, Waltham, MA) was added and incubated for 1 min. Thereafter, grids were wicked dry using qualitative filter paper (VWR, Radnor, PA), washed twice with 10 μl MilliQ water and then stained with 1% uranyl acetate for 2 min. Grids were wicked dry as above and allowed to air dry for at least 10 min, stored at room temperature and then examined using a 1200EX microscope (JEOL).
For measurement of soluble protein aggregates, at least three grids per protein were mounted, and at least three images per protein were analyzed. Length, defined as the long axis of each fragment measured, was measured using FIJI [77 (link)] and binned into 8 nm segments [66 (link)] using the scale bar as a standard (2.58 pixels/nm). Particles were excluded from analysis if they were on the border of the image or did not have clearly defined edges (i.e. due to a clustering). All particles meeting these criteria within the image field of view were also analyzed for width, defined as the short axis of each measured fragment.

TEM images of the thin-sectioned cells treated with polyelectrolytes (PAH 70 kDa, PEI, PDADMAC average Mw 200,000–350,000 (IC₅₀), and P(AAm-co-DADMAC) (100 µg/10⁵ cells)) were obtained using a 1200 EX microscope (JEOL, Tokyo, Japan) operating at 80 kV. The specimens were fixed with 2.5% glutaraldehyde, gradually dehydrated using a series of ethanol solutions (10–96%), embedded into Epon resin, and thin sections were cut using a ultramicrotome (LKB, Wetzlar, Germany) equipped with a diamond knife and mounted on copper grids. The thin-sectioned cells were stained using 2% aqueous uranyl acetate and lead citrate solutions.

Transmission electron microscopy investigations were carried out using a 1200 EX microscope fitted with link elemental dispersive X-ray analysis detectors and a 3010 UHR operating at 300 kV (JEOL Ltd, Tokyo, Japan). The powdered samples were ultrasonically dispersed in ultrapure water and a few droplets of the slurry were then deposited on perforated carbon foils supported on conventional copper microgrids. Scanning electron microscopy observations were carried out using an 840A microscope (JEOL Ltd). The specimens were mounted on aluminum stubs using carbon tape and covered with a coating of Au-Pd approximately 10 nm thick using a coating unit (Polaron Sputter Coater E5100, Polaron Equipment, Watford, UK).