Ex vivo imaging and enzymatic analysis of intestinal mucus.

The intestinal mucus layer is a highly hydrated hydrogel that forms a critical barrier separating the gut microbiota from the epithelial surface while permitting nutrient exchange and luminal transport. Despite its central role in intestinal homeostasis, native mucus remains difficult to study due to its transparency, fragility, and poor preservation by conventional fixation methods, which often distort its structure or detach it from the epithelium. These limitations have restricted investigation of mucus organization, barrier properties, and enzymatic processes under physiological conditions. Here, we describe experimental approaches for the visualization and functional analysis of intestinal mucus using live ex vivo explant preparations combined with confocal microscopy. This system preserves the native architecture, cellular heterogeneity, and mucus attachment of intact epithelial tissue while allowing controlled experimental manipulation. Fluorescent lectin-based labeling enables resolution of spatially distinct mucus domains, including crypt-associated plumes and intercrypt mucus, which differ in glycosylation patterns and penetrability. Functional barrier properties are assessed using bead penetration assays, providing quantitative measures of mucus thickness and resistance to particle infiltration. In addition, the use of fluorescent probes and peptide substrates permits detection and spatial mapping of endogenous and microbial enzymatic activity within the mucus layer. These methods enable investigation of how enzymatic remodeling influences mucus structure and barrier integrity. Together, these protocols provide a versatile platform for studying mucus dynamics, organization, and function in a physiologically relevant context, bridging the gap between in vitro systems and in vivo studies.