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). .