Three-dimensional Quantification of Intestinal Mucus Using Whole-mount Tissue Imaging

Mucus plays a critical role in maintaining intestinal homeostasis by facilitating digestion, forming a barrier against microbes, and regulating immune responses within the gut. Its secretion is modulated by various intrinsic and extrinsic factors, including microbial infections and inflammation. While traditional methods for mucus analysis rely on relative quantification, such as measuring mucus thickness in histological sections, these approaches provide limited insight into the actual volume and spatial distribution of secreted mucus within the intestinal lumen. Here, we present a detailed methodology for the absolute, three-dimensional quantification of mucus using whole-mount intestinal tissues and multi-photon microscopy. This protocol includes the preparation of ligated intestinal loops, treatment with carbamoylcholine chloride as a stimulus for goblet cell activation, tissue fixation, and staining for imaging. Using multi-photon microscopy, we acquire Z-stacked images of the luminal surface. These are processed in Imaris software to quantify the volume and spatial distribution of secreted mucus. We demonstrate that carbamoylcholine chloride robustly induces mucus secretion in both ileal and colonic tissues within 30 minutes. The secreted mucus appears in a sporadic and discontinuous manner throughout the lumen, underscoring the importance of volumetric analysis over traditional two-dimensional approaches. This protocol enables researchers to obtain absolute quantitative data and visualize mucus distribution in situ, offering a powerful tool for studying intestinal mucus dynamics under physiological and pathological conditions.