Azidosugar Labeling of Parasites

HFFs, grown on circular cover glass no. 1.5, were infected with either PLE-expressing or wild-type parasites for a day. The medium was replaced with complete medium with azidosugars or dimethyl sulfoxide. After a 2-day incubation with the azidosugars, the cells on a cover glass were fixed with 4% paraformaldehyde in PBS for 30 min and then permeabilized with 0.2% Triton X-100 for 20 min. The cells were incubated in 50 μM CuSO₄, 100 μM 3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]propanol (BTTP) (22 (link)), 50 μM Alexa Fluor 488–alkyne (Life Technologies), 2.5 mM sodium ascorbate, and 1% FBS in PBS for 20 min at room temperature. The cells were subsequently washed and immunostained with rabbit polyclonal anti-BAG1 antibody at 1:500, anti-CST1 antibody at 1:500, or biotinylated VVA lectin (Vector Laboratories) at 1:100 for 1 h at room temperature. The antibodies and the lectin were visualized by incubation with goat anti-rabbit–Alexa Flour 594 antibody at 1:2,000 or streptavidin-594 at 1:2,000 for 1 h at room temperature. The cover glass was then mounted on glass slides with the antifade.