Ultrastructural Analysis of Ileum Tissue

Samples were treated as previously described (Palmer et al., 2017 (link)). Tissues from the terminal ileum were cleared by flushing with HBSS (with calcium) at 37^o C and then fixed by vascular perfusion with 2% glutaraldehyde in Gomori phosphate buffer containing 0.1 mM EGTA (pH 7.4) at 37°C. Tissues were excised and cut along the antimesenteric plane, pinned flat in fixative for 10 min at RT, cut into small pieces, and then fixed o/n at 4°C in the same fixative. Tissues were washed in PBS, postfixed in 1% osmium tetroxide in Gomori phosphate buffer pH 7.4 for 1 h at 4° C, washed in distilled water, and then incubated o/n in 2% aqueous uranyl acetate at 4°C. After washes with distilled water, tissues were dehydrated in ethanol (Sigma-Aldrich, Natick, MA) and propylene oxide (Ted Pella, Redding, CA) prior to resin embedding in LX112 resin (Ted Pella). Ultrathin sections were cut with a Leica Ultracut E ultramicrotome (Leica Microsystems, Wetzlar, Germany), placed on formvar and carbon-coated slot grids, and then contrast stained with 2% uranyl acetate and lead citrate. DMs from cells along two villi from each mouse were imaged using a JEOL 1400 electron microscope (JEOL USA, Peabody, MA) equipped with an Orius SC1000 digital CCD camera (Gatan, Pleasanton, CA).