Uea1 fitc

Mice distal colons were sectioned (5 µm) after fixing in 4% paraformaldehyde. For histopathology analysis, sections were stained with hematoxylin and counter stained with eosin. Histological scoring of colon sections was evaluated by a blinded pathologist following these parameters: loss of epithelium (0–3), crypt damage (0–3), depletion of goblet cells (0–3), and inflammatory cell infiltrate (0–3). For immunostaining, human biopsy and mice colon samples were sectioned and after fixing with 4% paraformaldehyde antigen retrieval was performed. The sections were probed with anti-Rab7 (Sigma, R4779, 1:400), UEA1-FITC (Vector Laboratories, FL-1061, 1:400), anti-Muc2 (Santa Cruz, sc-515032, 1:200), and anti-CLCA1 (Abcam, ab180851, 1:100) in blocking (5% goat serum) overnight. Sections were incubated with HRP or fluorophore tagged secondary antibodies for 2 hr. Nucleic acid was stained with DAPI (1 µg/ml). Sections were cured and mounted with Prolong Gold Antifade Reagent (Thermo Fisher). Sections were imaged in confocal microscope (Leica SP8).

Thymic tissues were embedded in OCT compound (Sakura) and snap frozen in liquid nitrogen. 8 μm cryosections were prepared, air-dried and fixed for 10 min in cold acetone. Cryosections were blocked for 1 h at room temperature in PBS containing 5% FBS and 1 mg/ml anti-FcγRII/CD16 (2.4G2) (in-house production) before staining with UEA-1-FITC (Vector Laboratories). Images were taken on a confocal microscope (Zeiss LSM-710) and analyzed with ZEN 2010 software (Carl Zeiss, Inc.) and Fiji ImageJ. To measure the surface area of thymic epithelial cells, frozen thymic sections were stained with UEA-1 and Ly51 and high resolution pictures were obtained as described above. The size of UEA-1+ area and Ly51+ area in each thymic section was quantified by ImageJ separately and statistical analysis was done by GraphPad Prism 6 with a two-tailed Student’s t-test.