Goat anti rabbit igg

Cells were fixed for 2 h with 4% paraformaldehyde/0.1% glutaraldehyde in phosphate buffer (pH7.6), washed with PBS (pH7.6) for 2 × 5 min, and centrifuged at 500g for 10 min. After removing the supernatant, cell pellets were included in gelatin 12% and infused with sucrose 2.3 M overnight at 4 °C. 90 nm ultrathin cryosections were made a −110 °C on a LEICA UCT cryo-ultramicrotome. Sections were retrieved with Methylcellulose 2%/Sucrose 2.3 M mixture (1:1) and collected onto formvar/carbon-coated nickel grids. After removal of gelatine at 37 °C, sections were incubated on drops of 1:100 anti-Flag (F7425, Sigma). After six washes of 5 min each, grids were incubated on drops of PBS containing 1:30 gold-conjugated (10 nm) goat-anti-rabbit IgG (Aurion). Grids were finally washed with six drops of PBS (5 min each), postfixed in 1% glutaraldehyde, and rinsed with three drops of distilled water. Contrasting step was performed by incubating grids on drops of uranyl acetate 2%/methylcellulose 2% mixture (1:10). The sections were imaged in a transmission electron microscope at 100 kV (JEOL 1011).

Formvar/carbon-coated nickel grids were deposited on a drop of purified, unfixed viruses produced in the presence or absence of Flag-tagged IFITM3 for five minutes prior to sequential incubation with an anti-Flag antibody (F7425, Sigma, St-Louis, MO), followed by incubation with a 1:30 gold-conjugated (10 nm) goat-anti-Rabbit IgG (Aurion, Wageningen, Netherlands) and fixation in 1% glutaraldehyde. Negative staining was performed using 2% uranyl acetate (Agar Scientific, Stansted, UK) followed by, transmission electron microscope analyses (JEOL 1011, Tokyo, Japan).

Nickel grids coated with Formvar/carbon were deposited on a drop of purified, unfixed samples for five minutes prior to sequential incubation with an anti-Flag antibody (F7425, Sigma, St-Louis, MO, USA), followed by incubation with a 1:30 gold-conjugated (10 nm) goat-anti-Rabbit IgG (Aurion, Wageningen, The Netherlands) and fixation in 1% glutaraldehyde. Negative staining was performed using 2% uranyl acetate (Agar Scientific, Stansted, UK) prior to transmission electron microscope analyses (JEOL 1011, Tokyo, Japan).