Ccd camera

HNPs (1 mg/ml) in a volume of 5 µL were placed on a grid (Nisshin EM, Tokyo, Japan) and dried with a stream of warm air 3 times. Then, each sample was negatively stained with 10 µL of 1 w/v% ammonium molybdate for 1 min and imaged with an HT7700 TEM System (Hitachi High-Technologies, Tokyo, Japan). The images were recorded with a CCD camera at 1024 × 1024 pixels (Advanced Microscopy Techniques, Woburn, MA, USA).

The construction of VLPs was conducted as described in the previous study [11 (link)]. VLPs were based on a recombinant Baculovirus (rBV) expressing system using the HPAI H5N1 virus (A/chicken/Egypt/121/2012) [15 ]. In this study, we additionally explore the morphology and the size of the generated VLPs using Electron microscopy. VLP samples were adsorbed onto freshly discharged 400 mesh carbon parlodion-coated copper grids (Poly-Sciences, Warrington, PA USA). The grids were rinsed with buffer containing 20 mM Tris, pH 7.4, and 120 mM KCl and negatively stained with 1% phosphotungstic acid, then dried by aspiration. VLPs were visualized on a Hitachi H-7600 transmission electron microscope (Hitachi High Technologies America, Schaumburg, IL USA) operating at 80 kV and digitally captured with a CCD camera at 1 kx1 k resolution (Advanced Microscopy Techniques Corp., Danvers, MA, USA) [10 (link)].

Immunoelectroscopic analysis was performed essentially as described elsewhere (Hojo et al., 2004 (link); Mukai et al., 2007 (link); Ooishi et al., 2012b (link)). Rat hippocampus was frozen and sliced coronally. Freeze substitution and low-temperature embedding of the specimens was performed as described previously (Roberson et al., 1999 (link)). The samples were immersed in uranyl acetate in anhydrous methanol (-90°C). The samples were infiltrated with Lowicryl HM20 resin (Electron Microscopy Sciences, USA) and polymerization was performed with ultraviolet light. Ultrathin sections were cut using a Reichert-Jung ultramicrotome. For immunolabeling, sections were incubated with primary antibody for GR (Morimoto et al., 1996 (link); diluted to 1/3000) overnight, and incubated with secondary gold-tagged (10 nm) Fab fragment in Tris buffered saline (TBS). Sections were counterstained with 1% uranyl acetate, and viewed on a JEOL 1200EX electron microscope (Japan). Images were captured using a CCD camera (Advanced Microscopy Techniques, USA). The antibody is specific to GR in the hippocampus as shown with Western blot (Komatsuzaki et al., 2005 (link); Ooishi et al., 2012b (link)).