Visualizing Intestinal Mucus Using WGA

Intestinal mucus was visualized on-chip using wheat germ agglutinin (WGA)-Alexa 488 (ThermoFisher).⁵ (link) Briefly, 25 μg/mL of WGA in Hanks’ Balanced Salt Solution (HBSS, Gibco) was flowed at 200 μL/h through the apical channel for 30 min and then washed with continuous flow of HBSS at the same flow rate for 30 min. The entire channel was visualized with a fluorescence microscope (Excitation/Emission = 488/523 nm, Zeiss Axio Observer Z1). The mucus layer was set as the distance between the apical cell surface and the upper limit of the WGA-labeled material; the thickness of the mucus layer was measured by ImageJ. For MUC2 visualization, immunofluorescence microscopic imaging was carried out on 150–200 μm chip sections using antibodies against MUC2 (Santa Cruz, sc-15334) and a secondary antibody (ThermoFisher, A31573).¹² (link)^,13