Uea1 dylight647 conjugated lectin

Intestinal tissues containing luminal content were fixed by submersion in methanol-Carnoy solution for at least 24 h, and fixed tissue was paraffin embedded and cut into 5 µm thick longitudinal sections. Tissue sections were deparaffinised by sequential washing in xylene substitute (20 min at 60°C; Merck) and 100% (5 min), 95% (5 min), 70% (5 min), and 30% (5 min) ethanol. For histochemical staining, tissue sections were stained with Alcian blue and Periodic acid-Schiff (AB/PAS) stains as previously described.^{8 (link)} For fluorescent staining, antigen retrieval was performed by immersion of sections in 10 mM citrate buffer (95°C, 30 min). Sections were washed in PBS, permeabilized for 5 min using 0.1% vol/vol Triton X-100 (Merck), and blocked using 5% vol/vol FCS. To detect C. rodentium ICC169, sections were incubated overnight at 4°C with rabbit anti-O152 primary antibody (1:100, Denka Seiken). Sections were washed in PBS and stained with goat anti-rabbit Alexa 488-conjugated secondary anti-bodies (1:2,000; ThermoFisher) for 2 h at room temperature. Lastly, slides were washed with PBS and counterstained with a mixture of Hoechst DNA dye (5 µg/mL; Merck) and UEA1-DyLight647 conjugated lectin (10 µg/mL, Vectorlabs) for 15 min. Slides were rinsed in dH₂O, coverslipped using ProLong Gold Antifade mountant (Thermofisher) and imaged using an LSM700 confocal microscope (Zeiss).