Immunohistochemical Analysis of Bladder Tumor Markers

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).