Azide-Alkyne Cycloaddition for O-Glycan Detection

Detection of the azide labeled O-glycans was performed by conjugating the azide to a fluorescently labeled alkyne in a copper-catalyzed azide-alkyne cycloaddition reaction^{24 (link)} as previously described^{18 (link)}. Fixed paraffin embedded sections were dewaxed, hydrated, and washed in PBS. Tissue sections were incubated with 10 µl of reaction mix from the tetramethylrhodamine (TAMRA) glycoprotein detection kit (Invitrogen) and incubated at 4 °C overnight. After washing in PBS, the samples were blocked in 5% (v/v) fetal bovine serum and stained with anti-MUC2C3 antiserum (against a C-terminal peptide)^{25 (link)} or anti-Muc17S2 (against a peptide in the SEA domain)^{26 (link)}. The specific antibodies were detected with an Alexa 488 conjugated anti-rabbit antibody (Invitrogen) and the sections were mounted using ProLong anti-fade mounting medium (Invitrogen). The images were acquired using a LSM 700 Axio Examiner.Z1 laser scanning confocal microscope, with Plan-Apochromat 20x/0.8 and 40x/1.3 Oil DIC objectives (Zeiss). The images were acquired and processed uniformly using the ZEN 2012 software (Zeiss).