Gold conjugated anti mouse igg

Parasites (2 × 10⁷) grown under different glucose conditions were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at pH 7.2 at RT, treated with 1% (w/v) osmium tetroxide, and dehydrated with increasing concentrations of ethanol. Samples were transferred to propylene oxide and then successively to propylene oxide/LR-White resin mixtures at ratios of 1:1 and 2:1, embedded in LR-White resin, and polymerized at 56 °C overnight. Thin sections (60 nm) were obtained and incubated overnight at RT with the Mα-TvLEGU-2pep (mouse, 1:10) primary antibody, washed, and incubated with gold-conjugated anti-mouse IgG (1:60 dilution using 15 nm gold particles) as a secondary antibody (Ted Pella Inc., Redding, CA, USA). For double labeling, the sections were incubated with Mα-TvLEGU-2pep/RαTvPFO50r and Mα-TvLEGU-2pep/RαrTvAtg8b primary antibody combinations (both at 1:10 dilution). gold-conjugated anti-mouse IgG (1:60 dilution; 15 nm gold particles) and anti-rabbit IgG (1:60 dilution; 30 nm gold particles) were used as secondary antibodies (Ted Pella Inc.). The samples were counterstained with uranyl acetate and lead citrate. Samples incubated with PI serum or only the secondary antibodies were used as negative controls. A JEOL JEM-1011 transmission electron microscope was used (JEOL Ltd., Tokyo, Japan).