7600 transmission electron microscope

BSCs were washed in PBS and then fixed by 4% paraformaldehyde/0.25% glutaraldehyde in PBS for 1 h and processed for pre-embedding immuno-electron microscopy as previously described [21 (link)]. In brief, BSCs were incubated in 0.1% sodium borohydrate in PBS for 10 min and blocked with a solution of 5% horse serum, 1% bovine serum albumin, and 0.2% cold water fish skin gelatin in PBS for 1 h. BSCs were incubated overnight at 4 °C with primary antibody—7F2 to tau phosphorylated at Thr205 [51 (link)]. BSCs were then incubated with biotinylated-conjugated goat anti-mouse secondary antibody and developed with diaminobenzidine using Vectastain ABC kit as previously reported [56 (link)]. Tissue was silver enhanced as described by Rodriguez [45 (link)] and then post-fixed with 2% glutaraldehyde and 2% osmium tetraoxide, and, following dehydration in graded EtOH, they were embedded in Epon. Ultrathin sections of BSCs were cut and then negatively stained with 1% uranyl acetate and visualized with a Hitachi 7600 transmission electron microscope.

ChPl tissue was freshly isolated from the brain ventricles of 4 weeks old Atp7b^-/- and wild type mice and fixed by immersion with EM grade 2% paraformaldehyde 1% glutaraldehyde 100 mM phosphate buffer (Sorenson’s) and 3 mM magnesium chloride pH 7.2 at 4°C. all subsequent steps were done at 4°C until 70% ethanol dehydration. Tissues were dissected carefully in fixative measuring no more than 2 mm³. Samples were rinsed in buffer containing 3% sucrose (3 X 15 min). Osmication was performed in 1.5% potassium ferrocyanide reduced 1% osmium tetroxide in 100 mM phosphate buffer, containing 3 mM magnesium chloride for 2 hrs at 4°C. Tissue was rinsed in 100 mM Maleate buffer (3 X 5 min) containing 3% sucrose, then en-bloc stained with 2% filtered uranyl acetate in the same buffer for 1 hr. Samples were dehydrated to 100% ethanol.
Propylene oxide (2 X 5 min) was used as a transition solvent, and the tissue was embedded with eponate 12 (Ted Pella, Redding CA) and finally cured in 60°C oven for 2 days, then sectioned to 60 nm using diamond microtome. EM images were collected using a Hitachi 7600 transmission electron microscope operated at 80.0 kV. Images were acquired at 6,000 to 20,000 × direct magnification, corresponding to 0.010744 μm/pixel to 0.003223 μm/pixel, respectively.

EV isolation and preparation from airway epithelial cell and macrophage conditioned media was performed as we previously described [20 (link)]. Briefly, conditioned media were collected 8 or 24 h post cigarette smoke exposure and centrifuged at 1000×g for 10 min to remove dead cells and cellular debris. The supernatant was then filtered with a 0.22- $\mu$ m Nalgene filter (Thermo Scientific). The resulting supernatant was centrifuged at 10,000×g for 30 min to remove any remaining debris and then ultra-centrifuged at 118,000 × g for 70 min at 4 °C with a fixed-angle rotor (Ti-70, Beckman Coulter, Brea, CA). The pellet was washed with sterile 1X PBS and subjected to another cycle of ultracentrifugation at 118,000 × g for 70 min at 4 °C. The supernatant was discarded and the pelleted EVs were carefully reconstituted in sterile 1X PBS or lysed in RIPA buffer (ThermoFisher Scientific, Carlsbad, CA). EV concentration and characterization were determined using Nano Sight-based EV technology. EV morphology was characterized by imaging on a HITACHI 7600 transmission electron microscope equipped with AMTV600 camera at the University of Florida Transmission Electron Microscopy core [20 (link)].

Hearts of anesthetized mice were perfused with relaxing buffer (0.15% sucrose, 5% dextrose, 10 mM KCl in 1x PBS) for 3 min and then perfused with fixation buffer (1% paraformaldehyde, 2% glutaraldehyde in 100 mM sodium cacodylate pH 7.4), then fixed overnight in fixation buffer and post-fixed in 1% OsO₄ for 2 h. Ultrathin sections of all tissues were counterstained with uranyl acetate and lead salts. Images were obtained using a 7600 transmission electron microscope (Hitachi) connected to a digital camera (AMT, Biosprint16).

To purify virus, 30 ml of cell culture supernatant was overlaid on 4 ml of 20% sucrose in TNE buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA) and 2 ml of 55% sucrose in TNE in an SW 28 tube (Beckman-Coulter, Brea, CA). Samples were spun for 3 h at 25 k RPM in an SW 28 rotor. Purified virus was collected from the 20%-55% sucrose interface, diluted with TNE and pelleted for 2 h at 55 k RPM in an SW 55Ti rotor. Pellets were resuspended in 40–60 μl TNE and kept on ice for immediate use.
Purified virus was treated with 8.0 × 10^-3 g/ml of S. nigra extract or 0.8% ethanol as a vehicle control in PBS for 15 min at room temperature. Samples were then spotted onto a glow discharged, carbon coated copper grid (Electron Microscopy Sciences, Hatfield, PA) and incubated for 2 min. Grids were rinsed with water, and stained for 1 min with 2% phosphotungstinic acid, pH 7.4. Samples were examined on a Hitachi 7600 transmission electron microscope under 80 kV, and micrographs collected using AMT Image Capture Engine software controlling an AMT ER50 5 megapixel CCD camera (Advanced Microscopy Techniques Corp., Danvers, MA).