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.
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.
