Muc2 sc 15334

The following antibodies (BioLegend) were used for flow cytometry: CD45 (30-F11), CD326/EpCAM (G8.8), CD44 (IM7), Sca-1 (D7), TCRβ (H57), γδTCR (GL3), NK1.1 (PK136), CD90.2 (53-2.1), CD11b (M1/70), Gr1 (RB6-8C5). For immunofluorescence staining in section, the follow antibodies were used: GFP (GFP-1020, Aves; ab13790, Abcam), Ki67 (Sp6, Thermo Fischer Scientific), E-cadherin (24E10, Cell Signaling Technology), Sca-1 (e13-161.7, Biolegend), Muc2 (SC-15334, Santa Cruz Biotechnology), Mmp7 (AF2967, R&D Systems). EdU was detected using Click-iT Plus EdU Assay Kit (ThermoFisher).

Primary intestinal crypt cultures (enteroids) were established and maintained in culture according to the methods published by Sato et al. (11 (link)) with a few modifications (9 (link), 12 (link)). Enteroids were seeded on Matrigel, allowed to grow for at least 24 h before treatment with recombinant rat IL-17A (Peprotech – 100 ng/mL) or vehicle alone for 6 hours in order to evaluate cell proliferation, differentiation and death by immunohistochemistry (IHC) and confocal microscopy as described in the next section. The following antibodies were used for IHC analysis: BrdU (BRD494 – Novus Biosciences), chromogranin A (ab15160 – Abcam), e-cadherin (AF748 – R&D Systems), Ki67 (ab15580 – Abcam) and mucin glycoprotein 2 (Muc2, sc-15334 – Santa Cruz). Nuclear counterstaining was performed using DAPI (Pierce).
All-trans retinoic acid (ATRA) was obtained from Sigma-Aldrich, dissolved in DMSO and corn oil (1:1 – final concentration 6 mg/mL, protected from light) and administered daily by gavage to breast-fed and NEC mice (50 μg/mouse) for the duration of the experimental induction of NEC.