Biotinylated vva lectin

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.

FFPE bladder tumors and healthy tissue sections were screened by immunohistochemistry for CD44 and Tn and STn antigens, as previously described by Peixoto, A et al. 31 (link). Tn antigen expression was evaluated using the biotinylated VVA lectin (Vector Laboratories, 40 mg/mL, 1 hour at 37 ºC; Table S2) and the detection of STn and CD44 antigens were performed using the anti-tag-72 (B72.3 + CC49; Abcam, 0.5 mg/mL, overnight at 4 ºC; Table S2) and anti-CD44 (1:5000, ab157107, Abcam; Table S2) antibodies, respectively. Lack of cross-reactivity of VVA for blood group A and AB antigens was confirmed using the anti-blood group A monoclonal antibody (HE-193, Thermo Fisher, 1:5, overnight at 4 ºC; Table S2). Sialidase treatment of tissue samples prior to anti-STn probing was also performed to confirm the presence of the glycan (Sigma-Aldrich, 0.2 mg/mL, overnight at 37 ºC). CD44 and anti-tag-72 were detected using Novolink Polymer Detection System (Leica) according to manufacturer guidelines. Biotinylated VVA was detected using Streptavidin, Horseradish Peroxidase Conjugate (Thermo Fisher, ready-to-use, 30 min, RT) followed by incubation with ImmPACT® DAB Substrate, Peroxidase (Vector, 30:1000, 5 min, RT). All images were acquired on a Motic BA310E microscope (Motic) using the Motic Images Plus 3.0 software (Motic).