Immunogold Labeling of Vimentin in Ultrathin Sections

Ultrathin LR-White embedded sections, collected on Formvar carbon-coated nickel grids, were incubated in drops of 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) containing 0.02 M glycine and normal goat serum at room temperature for 30 min [38 (link)]. Sections were then incubated overnight with a rabbit monoclonal anti-vimentin antibody (clone V9; Ventana, Tucson, AZ, USA; pre-diluted) at 4°C. After several washes with PBS + 0.1% BSA, grids were incubated with a 20 nm secondary antibody-gold particle complex (Agar Scientific, Stansted Essex CM24 8GF United Kingdom) at 1:10 diluted in PBS 0.1% BSA for 2 h at room temperature. After immunolabeling, sections were washed with PBS + 0.1% BSA, washed in distilled water, dried, and counterstained with uranyl acetate. All sections were examined with a Hitachi 7100 FA electron microscope.