Orius sc200b ccd camera

After the immersion fixation, the optic nerves from each animal (n = 7–9) were incubated at 4 °C until further washing with 90 mM Na-cacodylate buffer with subsequent processing as described previously [20 (link)]. Electron micrographs for analysis were collected using an Orius SC200B CCD camera (Gatan Inc., Warrendale, PA, USA) mounted on a of Tecnai G² Spirit TWIN/BioTWIN transmission electron microscope (FEI Europe B.V, Eindhoven, The Netherlands). Representative figures for illustrations were collected using a side mounted Veleta CCD camera (Olympus).

Following PBS perfusion, mouse brainstem tissue was dissected and frozen in liquid nitrogen. Sarkosyl extraction was performed as previously described (Delobel et al., 2008 (link)). Briefly, the brainstem tissue was homogenized in A68 buffer (0.5 ml of 800 mM NaCl, 10% sucrose, 10 mM Tris-HCl pH 7.4, 1 mM EGTA) using a Kinetica polytron. Samples were centrifuged at 5000g for 15 min. The collected supernatant was analysed as total tau samples. Following sarkosyl addition to 1% and shaking for 1 h, samples were centrifuged at 80 000g for 30 min. The resulting pellet was resuspended in 50 mM Tris-HCl pH 7.4.
For immunoelectron microscopy, aliquots were placed on carbon-coated 400 mesh grids and allowed to dry partially. Grids were blocked in droplets of 0.1% gelatin (Sigma G7041, Sigma-Aldrich) and stained with HT7 primary antibody (1:50; Pierce). Grids were then washed briefly with blocking buffer, stained with anti-mouse IgG-Gold secondary antibody (Sigma G7652), washed with water, and stained with 2% uranyl acetate. Electron microscopy was performed using a FEI Tecnai Spirit TEM at a magnification of ×21 000 and images recorded using a Gatan Orius SC200B CCD camera (Gatan).

The study was performed as described previously.18, 24 Vehicle control or TN16‐treated cells were fixed overnight at 4°C using 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) followed by osmication and encapsulation in agarose by centrifugation. Approximately 1 mm³ pieces of pellet were cut and subjected to dehydration in ascending series of ethanol and acetone, before finally being embedded into Spurr resin. Sixty to eighty nanometre thick sections were obtained Leica EM UC7 microtome which were collected over 200 mesh copper grids. The sections were double‐stained with uranyl acetate and lead citrate and air‐dried before viewing under JEOL JEM 1400 transmission electron microscope at 80kV using Gatan Orius SC200B CCD camera.