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

Our research focuses on the mechanisms of microbial injection with mucosal tissues, emphasizing the pivotal role of mucus in modulating these interactions. Traditional 2D mucus quantification misses spatial and volumetric details. Our protocol enables accurate 3D analysis, revealing structural and distributional changes by molecular interventions overlooked in existing methods.

This protocol combines hormone imaging with 3D quantification, providing robust reproducible measurements of intestinal mucus architecture applicable to diverse experimental conditions and molecular interventions surpassing conventional 2D assessment. To begin, place an anesthetized six to eight week old mouse on a heating pad, to maintain its body temperature. Disinfect the skin surface of the mouse with alcohol, and make an incision in the left lower abdomen to expose the cecum while maintaining anesthesia.

Now, identify the ileum or proximal colon. Place the intestinal loop on sterile gauze. Rinse with sterile PBS and secure both ends with arterial clamps to create a three-centimeter-long closed loop.

Under anesthesia, inject no more than 200 microliters of sterile PBS or carbamoylcholine chloride reagent into the ligated intestinal loop. After 30 minutes of treatment, harvest the intestinal loop from the euthanized animal. Using forceps, hold the intestinal loop and gently rinse it in sterile PBS.

Cut open the intestinal loop longitudinally, with a small uncut portion, and rinse the tissue again in sterile PBS. Flatten the tissue in a six-centimeter dish and cut open the remaining portion. Use copper wire segments to anchor it onto the agarose base.

Now, add 10 milliliters of 4%paraformaldehyde solution to the dish to fix the tissue for 12 to 24 hours. After incubation, remove the paraformaldehyde solution and wash the tissue at least three times with PBS to remove any residual paraformaldehyde. Next, immerse the tissue in 10 milliliters of blocking buffer consisting of PBS supplemented with 0.4%Triton X-100 and 3%BSA, and stored at four degrees Celsius for 12 to 24 hours.

Remove the blocking buffer. Stain the tissue with dye solution containing wheat germ agglutinin and phalloidin. In PBS-treated small intestine and colonic tissues, WGA-positive mucus was predominantly localized within goblet cells at the epithelial surface.

Following carbamoylcholine chloride treatment, the amount of WGA-positive mucus in the intestinal lumen increased noticeably in both the small intestine and colon, but the mucus was dispersed sporadically rather than forming a continuous layer. The volume of goblet cell associated WGA-positive mucus was significantly reduced after carbamoylcholine chloride treatment in both the small intestine and colon, indicating active secretion. All goblet cell mucus volumes in PBS-treated tissues were below 1, 000 cubic micrometers.

WGA-positive items exceeding 1, 000 cubic micrometers were classified as secreted mucus in the lumen. Carbamoylcholine chloride treatment increased the total volume of secreted mucus by approximately five to 20 times in both small intestine and colonic tissues.