Mouse monoclonal anti ha antibody

TriEx Sf9 cells co-infected with recombinant baculoviruses containing PRRSV M, N, E, and GP5 gene were harvested at 72 h after infection. Cells were lysed with a lysis buffer containing 0.01 M Tris-HCl, 0.14 M NaCl, 0.025% NaN3, 1% Triton X-100, and protease/phosphatase inhibitors cocktail (Thermo Scientific, Rockford, IL). Cell lysates and purified VLPs were subjected to SDS-PAGE gel electrophoresis (Novex by Life Technologies, Carlsband, CA) and transferred to a nitrocellulose membrane (Life Technologies, Gaithersburg, MD). The membrane was then blocked for 1 h by rocking slowly at room temperature with 5% (w/v) milk powder in PBS plus 0.05% Tween 20 (PBST). Next, primary antibody, monoclonal mouse anti-HA antibody (Sigma-Aldrich, St. Louis, MO) diluted 1:5,000 in blocking buffer 5% (w/v) milk powder in PBST was added to the membrane and incubated overnight at 4 °C on the rocker. The secondary antibody, goat anti-mouse IRDye (LI-COR, Lincoln, NE) diluted 1:10,000 in PBST was added to the membrane and incubated for 1 h rotating at room temperature. Bands were visualized using the ODESSY Infrared Imaging System (LI-COR, Lincoln, NE).

Ultrathin (70–90 nm) sections were collected on nickel slot grids as described by ref. 50 . For immunogold labeling, sections were blocked for 30 min in a 1:30 dilution of goat normal serum in TRIS buffer (20 mM TRIS, 15 mM NaN3, 225 mM NaCl, pH 6.9) supplemented with 1% (w/v) BSA (Sigma-Aldrich A3294) and 1% (w/v) fish gelatin (FG; Sigma-Aldrich G7765). After three washes for 10 min in TRIS-BSA-FG, sections were incubated in a 1:500 dilution of monoclonal mouse anti-HA antibody (Sigma Aldrich H-9658) for 1 h at room temperature (slow orbital shaking). After washing in TRIS-BSA-FG (4× 10 min), sections were incubated in a 1:20 dilution of secondary goat anti-mouse antibody conjugated to 10 nm colloidal gold particles (British Biocell International, Cardiff, UK) for 1 h (slow orbital shaking). Finally, sections were rinsed in TRIS-BSA-FG (4 × 5 min) followed by a stream of sterile-filtered, distilled water for 3 min. After drying at room temperature, sections were examined without further staining in a Hitachi H-7650 TEM (Hitachi High-Technologies Europe GmbH, Krefeld, Germany) operating at 100 kV fitted with an AMT XR41-M digital camera (Advanced Microscopy Techniques, Danvers, USA).