Fei tecnai 12 transmission electron microscope

TA muscles were longitudinally stretched and fixed overnight at 4°C with 2.5% glutaraldehyde (Sigma-Aldrich) and 2% formaldehyde (Sigma-Aldrich) in 0.1 M sodium cacodylate buffer pH 7.4, and postfixed for 2 h at 4°C with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer. After three water washes, samples were dehydrated in a graded ethanol series and embedded in an epoxy resin (Sigma-Aldrich). Ultrathin sections (60–70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate. Images were acquired with a FEI Tecnai 12 transmission electron microscope (FEI Company) operating at 100 kV and equipped with a Veleta CCD digital camera (Olympus Soft Imaging System).

Infected cells on 3 mm sapphire glass coverslips were frozen in a Bal-tec HPM010 HPF according to [34 (link)] before being transferred to a pre-cooled (−160 °C) automatic freeze substitution unit (AFS, Leica Microsystems, Milton Keynes, UK) and allowed to warm to −90 °C over a period of 3.5 h. FS protocol has been described elsewhere [34 (link)]. Briefly, cold (−90 °C) freeze substitution medium containing 0.2% uranyl acetate in acetone was carefully added to the samples and left for 60 min before warming to −50 °C. Samples were taken through a graded series of resin concentrations (Lowicryl HM20, Agar Scientific, Stansted, UK, in acetone), placed in Leica flat embedding moulds (cells facing upwards) and polymerized under UV light at −50 °C for 48 h and room temperature for a further 48 h. Thin sections (60 nm) of the resulting resin blocks were cut (Leica Microsystems, UC6 ultramicrotome, Milton Keynes, UK) and imaged at 100 kV in a FEI Tecnai12 transmission electron microscope (Thermo Fisher Scientific, Waltham, MA, USA) with Tietz F216 2K × 2K CCD camera (TVIPS, Munich, Germany) after counter staining with uranyl acetate and lead citrate (EM Stain, Leica Microsystems, Milton Keynes, UK).

Corneas were processed for serial block-face scanning electron microscopy (SBF-SEM) as described previously in detail (24). Briefly, the corneas were fixed in 0.1 M sodium cacodylate buffer containing 2.5% glutaraldehyde, post-stained with heavy metals (Fe, OsO4, uranyl acetate, lead) before dehydration through an acetone series and embedding in Embed 812 resin (Electron Microscopy Sciences, Hatsfield, PA, USA) containing Ketjenblack EC600JD (Lion Specialty Chemicals Co., Tokyo, Japan). The resin-embedded blocks were sputter-coated with gold to reduce charging during block-face imaging. Tissue blocks were sectioned at 100 nm using a Gatan 3View2 system (Gatan, Pleasanton, CA, USA) mounted to a Mira 3 scanning electron microscope (Tescan, Pittsburgh, PA, USA). In some cases, platelets within the injured cornea were immunogold labeled and imaged using an FEI Tecnai 12 transmission electron microscope (ThermoFisher, Sugarland, TX, USA). These corneas were initially fixed with phosphate-buffered saline (PBS) containing 2% paraformaldehyde, permeabilized with 0.1% Triton-X 100 and sequentially labeled with a platelet-specific primary rat anti-CD42b antibody (10 µg/mL; InVitrogen, ebioscience, Carlsband, CA, USA) followed by a secondary goat-anti-rat IgG antibody conjugated to 5 nm gold particles (Nanoprobes, Yaphank, NY, USA).

VLPs were subjected to immunogold labeling. The procedures were adapted from a previous published method [38 (link)]. A total of 4 μL of diluted samples was incubated on glow-discharged EM grids (FCF300-Ni; Electron Microscopy Sciences, Hatfield, PA, USA) for 5 min at room temperature. The grids were incubated on droplets of 20 mM glycine for 10-min, followed by a 30-min blocking in 5% bovine serum albumin in PBS without calcium chloride, without magnesium chloride (DPBS, [−] Ca, [−] Mg). The grids were floated onto 30 μL droplets of palivizumab (1:50) or Motavizumab (40 μg/mL) in 5% BSA in DPBS ([−] Ca, [−] Mg) overnight at 4 °C. After three washes with 1% BSA in DPBS ([−] Ca, [−] Mg), 5-min per wash, the grids were placed on 30 μL of droplets of 6-nm gold beads conjugated with goat anti-human polyclonal IgG antibody (Electron Microscopy Sciences, Hatfield, PA) at 1:100 dilution in 5% BSA in DPBS ([−] Ca, [−] Mg), for 1-h at room temperature. Then the grids were washed three times with 1% BSA in DPBS ([−] Ca, [−] Mg), and stained with 1% PTA (pH 6~7), as described above. The negative-stain grids were imaged in low-dose mode (50 e⁻/Å), using a FEI Tecnai 12 transmission electron microscope (Thermo Fisher Scientific, previously FEI, Hillsboro, OR) at 120 kV, images were acquired on a 4k × 4k Gatan OneView Camera (Pleasanton, CA, USA).