Ultrascan 1000 ccd camera

Worms were high pressure frozen in either a Bal-Tec HPM 010 (Bal-Tec AG, Liechenstein) or Leica HMP 100 (Leica Microsystems, Vienna) high pressure freezer and freeze substituted in 1% osmium tetroxide and 0.1% uranyl acetate in acetone over a period of 2 hours by the SQFS method of McDonald and Webb [45] (link). Infiltration of Epon epoxy resin was carried out by 15 minute incubations in 25, 50, and 75% acetone-resin mixtures on a rocker, then three 15 minute incubations in pure resin. Polymerization of resin was for 2 hours in a 100°C oven. Sections of 70 nm thickness were post-stained with 2% uranyl acetate in 70% methanol for 4 minutes and lead citrate (Reynolds, 1963) for 2 minutes. Images were viewed on a Tecnai 12 (FEI Inc., Hillsboro, OR, USA) transmission electron microscope operating at 120 kV, and images recorded with a Gatan Ultrascan 1000 CCD camera (Gatan Inc., Pleasanton, CA, USA). Some high magnification views of microtubule were taken out of focus in order to highlight protofilament patterns [48] (link), [49] (link).

Aggregates of P23T hγD formed under acidic (pH 3) and neutral (pH 7) conditions were imaged using TEM. Samples (5 μl) were adsorbed on freshly glow discharged 400 mesh size carbon-coated copper grids for 15 s, and the excess was removed by blotting with filter paper. The sample grids were subsequently stained with 1% (w/v) sodium phosphotungstate for 5 s, and blotted. Grids were imaged at 11,000-fold (pH 7) and 21,000-fold (pH 3) magnification using a Tecnai T12 transmission electron microscope (FEI; Hillsboro, OR) operating at 120 kV and equipped with an UltraScan 1000 CCD camera (Gatan; Pleasanton, CA).

WT and mutant B/Brisbane/60/2008 viruses in suspension were adsorbed to the surface of transmission electron microscope grids for 15 min. Grids were blocked with a blocking solution (Aurion Electron Microscopy Sciences) for 30 min followed by incubation with the control IgG, 34B5, 46B8 or 46B8 N297G for 1 h at room temperature. Grids were washed and incubated with a biotinylated donkey-anti-human secondary antibody (Jackson ImmunoResearch) for 1 h at room temperature. Grids were washed again and then incubated with 15 nm gold-conjugated streptavidin (Ted Pella) for 1 h at room temperature. Finally, grids were washed, treated with 4% paraformaldehyde to inactivate all virus particles and counter stained with 2% ammonium molybdate for 1 min. Samples were examined under a JEOL JEM-1400 transmission electron microscope at 120kV. Digital images were captured with a GATAN Ultrascan 1000 CCD camera at magnifications from × 1,000 to × 50,000.

For transmission electron microscopy, first trimester placenta was fixed in 0.1 M phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% formaldehyde (2 h), post-fixed in 2% OsO4 (2 h), dehydrated in graded series of ethanol, and embedded in a TAAB epoxy resin (Gröpl). Ultrathin sections (75 nm) were cut with a Leica UC 7 and stained with lead citrate and platine blue. Images were taken on a FEI Tecnai G2 20 transmission electron microscope (FEI, Eindhoven, the Netherlands) with a Gatan ultrascan 1000 CCD camera (acceleration voltage 120 kV.