Orca hr camera

Cell supernatants containing HCoV-OC43 particles were concentrated by centrifugation at 39,000 rpm in an SW-41 rotor with an Optima™ L-90K Ultracentrifuge (Beckman Coulter) for 2 h. Pelleted virus was resuspended in 100 µL of PBS. A volume of 80 uL of the preparation was diluted to 400 uL with serum-free DMEM, divided equally into clear or black microtubes and treated with Phytoquin or DMSO vehicle control as described in Section 2.3 above. Small drops (~30 µL) were placed onto Formvar/Carbon coated grids and left to settle for 10 min before rinsing the grid with distilled water. The grids were quickly rinsed with a small drop (~30 µL) of 2% uranyl acetate then stained for 30 s in a second drop. The stain was then wicked off on filter paper and left to dry completely. Grids were imaged with a JEM 1230 transmission electron microscope (JEOL) running at 80 kV and an ORCA-HR camera (Hamamatsu). Sample preparation, fixation, and TEM imaging were performed twice with representative images from one independent experiment shown.

Pieces of intestinal (jejunum) tissues were excised and prepared for transmission electron microscopy (TEM) by fixing in 2.5% glutaraldehyde in 0.1 mM cacodylate buffer at room temperature for 1 h followed by overnight fixation at 4 °C. Samples were washed and incubated with 1% osmium tetroxide (pH 6.0) for 1 h, stained with 1% uranyl acetate overnight at 4 °C followed by ethanol dehydration (30%, 50%, 70%, 95%, and 100%). Samples were further dehydrated in propylene oxide. After dehydration, tissue pieces were infiltrated with and embedded in EM bed 812 resin (cat no. 14120; Electron Microscopy Sciences, Hatfield, PA, USA). After solidification, blocks were sectioned on the ultramicrotome at 60–70 nm setting (silver or silver-gold colored section appearance) and grids with sections were stained with 2% uranyl acetate for 20 min and Reynold’s lead citrate for 1 min at room temperature. Grids were examined under the electron microscope equipped with Hamamatsu Orca HR camera and AMT camera system, HV 80.0 kV.

E14.5 embryonic hearts were dissected and fixed for 30 min in 4% paraformaldehyde followed by a 1 hr wash in distilled water and secondary fixation overnight. Fixed samples were dehydrated and embedded in modified JB4 methacrylate resin (Weninger et al., 2006 (link)) and sectioned at 2 μm. HREM imaging (isometric resolution of 2 μm) used a Hamamatsu Orca-HR camera. Data sets were normalized and subsampled prior to 3D volume rendering using Osirix v5.6 (Rosset et al., 2004 (link)). As the expected CHD were not fully penetrant, a minimum of 17 E14.5 mutant embryos were compared to littermate controls. Phenotype analysis was performed blind for genotype, and classification of type of CHD was carried out as previously described (Dunlevy et al., 2010 (link)). For dual-wavelength HREM, a conventional Xgal reaction was performed followed by 4% paraformaldehyde fixation, dehydration and embedding, and imaging was carried out using a Jenoptik ProgRes C14 camera with dual filter (59022bs, Chroma Technology Corp). Image analysis of the DMP was done using ITKsnap 2.4.0 and Volocity 6.2.1 software packages. Volocity was used to calculate the volume of the DMP and the shape factor (shape factor is 1 for a perfect sphere and <1 for more irregular shapes). A minimum of 9 biological replicates per group was analyzed and analysis of the DMP was performed blind for genotype.

Brain sections from Bregma 2.92 mm to −3.52 mm were rinsed in PBS (50 mM, pH 7.4), postfixed flat in 1% osmium tetroxide, dehydrated in ascending concentrations of ethanol, treated with propylene oxide, and impregnated in Durcupan (Sigma-Aldrich, Oakville, ON, Canada) overnight at room temperature as described (Bisht et al., 2016a (link)). After resin polymerization at 55°C for 72 h, the areas of interest containing ventral HPC CA1 were cut at 70 nm with an ultramicrotome (Leica Ultracut UC7). Ultrathin sections were collected on square-mesh grids and examined at 80kV using a FEI Tecnai Spirit G2 microscope. For analysis, 16 pyramidal cells and 10 interneurons on average, in each of strata oriens and radiatum, were randomly photographed at magnifications between 890× and 4800× using an ORCA-HR camera (10 MP; Hamamatsu). Strata radiatum and oriens were identified based on their cellular and subcellular contents, and position relative to the CA1 pyramidal layer.