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On the 3day after the last FMD cycle was completed, mice were euthanized, and serum, spleen, small intestine, and big intestine (ceca and colon) tissue was collected. Serum and spleens were kept on ice for FACS-processing and cytokine measurements. Big intestines were straightened to measure colon length, and ceca contents were removed for the fecal transplant experiment. Small and big intestinal tissue was then cut and either flash-frozen and stored at −80°C or fixed in 4% PFA and further processed in a sucrose gradient, OCT-embedded, sectioned, and stained. The jejunum portion of the small intestine and the proximal colon of the big intestine were used for all staining procedures described here, and the tissue preparation described has been adapted from previous protocols (). Intestinal tissue was fixed in 4% PFA overnight, rinsed in 1X PBS and suspended in a 10% sucrose solution overnight. To complete the cryoprotection process, tissues were finally suspended in a 30% sucrose solution overnight. Tissues were then embedded in optical cutting temperature medium (OCT), flash-frozen on dry ice, and stored at −80°C until ready for cryo-sectioning. Sections were transversally cut at 7μm and adhered to positively-charged microscope slides. The exception to this procedure was for H&E staining: proximal colon sections were not fixed in 4% PFA and were immediately kept in 1X PBS after cutting open longitudinally, rolled with a toothpick, mounted in cryomolds using OCT, and flash-frozen on dry ice. Sections were transversally cut at 4μm and adhered to positively-charged slides that were stained with the Hematoxylin & Eosin Stain Kit (Vector Laboratories, H-3502) following the manufacturer’s protocol to visualize general changes in epithelial and crypt morphology. To quantify colonic crypt number for each animal, representative images from each section (3-4) of a slide were taken, and the distance in pixels was measured along the muscularis mucosae. The distance was converted to μm, and the number of crypts counted over the measured distance was normalized to a number over 5000 μm. For immunofluorescent staining of CD4, CD8, CD11b, Lgr5, Sox9, and BrdU, slides containing jejunum small intestine and proximal colon sections were thawed at room temperature, rehydrated with PBS, blocked for non-specific staining in blocking buffer (1% Normal Donkey Serum in PBS, Jackson ImmunoResearch) for 30 min at room temperature, and stained with primary antibodies overnight at 4°C. The next day, the sections were washed and stained with secondary antibodies for 1 hour at room temperature. Sections were protected from light, washed in PBS, stained with Hoechst 33342 (Thermo Fisher) to stain for nuclei, and coverslipped with anti-fading polyvinyl alcohol mounting medium with DABCO (Sigma-Aldrich). Images were captured at 20X using the EVOS FL Cell Imaging System (Thermo Fisher) and BZ-X710 All-in-One Fluorescence Microscope (Keyence) and analyzed with ImageJ (National Institute of Health). The quantification of CD4, CD8 and Sox9 positive cells was limited to the villi region for small intestine sections, and the number of positive cells in 30-50 villi per animal were counted (at least 50 for Sox9). The quantification of Lgr5 was localized to the crypt region for both the small intestine and colon samples, and the number of positive cells in 50 crypts per animal were counted. The quantification of CD4, CD8, and Cd11b for colon sections was limited to the LP surrounding the base of colonic crypts. The area surrounding 50 randomized crypts per animal were counted. The average number of positive cells per villi or crypt was then derived from these calculations. For BrdU positive cell detection in the crypts of small intestine jejunum and proximal colon tissue, sections were stained with BrdU In Situ Detection Kit (BD Biosciences, 550803) following the manufacturer’s 3,3′-diaminobenzidine (DAB)-based protocol. Images were observed with an Olympus BX50 microscope and Olympus DP73 camera and captured at 20X (colon) or 40X (small intestine) magnification with cellSens Standard (Olympus Corp.) software. The number of positive BrdU cells in 50 crypts per animal was counted, starting from the base of the crypt to two-thirds of the way up from the base. The average number of BrdU positive cells per crypt was then derived from these calculations. Primary antibodies used for small intestine and colon immunofluorescent staining: mouse anti-CD4 (eBioscience, 14-0041-86), rat anti-CD8 (Abcam, ab22378), rabbit anti-Lgr5 (Abcam, ab75732, 1:200), mouse anti-Sox9 (eBioscience, 14-9765-80). Secondary and secondary-conjugated primary antibodies used for small intestine and colon immunofluorescent staining: Alexafluor 488 anti-mouse/human CD11b (BioLegend, 101219), donkey anti-rabbit Alexafluor 594 (Thermo Fisher, A21207), donkey anti-mouse Alexafluor 488 (Thermo Fisher, A21202), and donkey anti-rat Alexafluor 594 (ThermoFisher, A21209).