Histology and immunohistochemistry
Dissected intestinal segments (jejunum, ileum,colon) were fixed in 4% paraformaldehyde and embedded in paraffin according to standard procedures. 3µm thick sections were stained with Hematoxylin and Eosin (HE) and basic histological parameters such as crypt-villus length, crypt depth and muscle layer thickness were assessed (Alshamy, Richardson, Hünigen, Hafez, Plendl & Al Masri, 2018). Crypt-villus length was defined as the distance from the crypt base to the apex of the villus in the jejunum and ileum. Crypt depth was measured from the basement of crypt to the surface epithelium in the proximal and mid-distal colon. Three to eight villi or crypts were measured and averaged for every section. Muscle layer thickness was measured from serosa to inner muscularis mucosae. Intestinal sections were also stained with Toluidine blue to detect mast cells numbers (Norkina, Kaur, Ziemer & De Lisle, 2004). Images were acquired under the Leica DFC295 light microscope. Mast cells were then counted in five different sections per segment. The results are expressed as cells per mm2 (image field area).
The dissected intestinal segments were processed for immunohistochemical (IHC) staining as previously described (Kini et al., 2020). The sections were incubated overnight at 4°C with primary antibodies to Muc2 (1:200, Santa Cruz Biotechnology, Heidelberg, Germany, Cat#sc-15334, RRID: AB_2146667) and MPO (1:250, Proteintech, Planegg-Matinsried, Germany, Cat# 22225-1-AP, RRID:AB_2879037). It was then incubated with the corresponding secondary antibody (Goat anti-rabbit Alexa 568; 1:500, Thermofisher, Waltham, MA, USA, Cat #A-11036, RRID AB_10563566) and DAPI to detect the nuclei. The slides were mounted with Mowiol 4-88 and images were acquired on the Olympus FluoView™ FV1000 confocal microscope. Muc2 was quantified as per previously described with a few modifications (Honda et al., 2017). Briefly, the intensity of green fluorescence indicating MUC2 stained mucins were quantified using Image J software. A region of interest (ROI) was determined (Supplementary Figure 1a) individually for intracellular and secreted mucins. Followed by normalizing the detected area with the background, the integrated density (I.D) ratio of Muc2 to Dapi was calculated for total, as well as for the intracellular and secreted mucins. The additive Muc2 to Dapi ratio of the intracellular and secreted mucins represents the total Muc2. MPO positive cells were counted by eye in image taken at 40X magnification in five different sections per segment. The results are expressed as cells per mm2 (image field area). .