Intestinal Epithelial Cell Subtype Profiling

Intestinal specimens were harvested and fixed in 4% paraformaldehyde prior to cryoprotection, OCT embedding and cryosectioning. Sections (10 μm) were stained using various IEC subtype-specific antibodies or lectins: rat-anti-E-cadherin (all IECs; ECCD-2, Takara, #M108, Mountain View, CA, USA), goat-anti-Anterior Gradient 2 (goblet cells; Santa Cruz, #sc54561, Dallas, Texas, USA), rabbit-anti-Chromogranin A (enteroendocrine cells; ImmunoStar, #20086, Hudson, WI, USA), Ulex europaeus lectin-FITC (Paneth cells; Sigma-Aldrich, #L9006, St. Louis, MO, USA). Secondary antibodies were donkey-anti-rat-AF594 (Life Technologies, #A21209 Carlsbad, CA, USA), donkey-anti-goat-Cy3 (Jackson Immuno Research, #705-166-147, West Grove, PA, USA) and donkey-anti-rabbit-BV421 (Nordic BioSite, #406410, Täby, Stockholm, Sweden). HOECHST (Life Technologies, #H1399) was used as DNA counterstaining. Slides were scanned using Metafer automated slide scanner (MetaSystems, Altlussheim, Baden-Württemberg, Germany) and composite pictures analyzed using the Visiopharm Integrator System program [16 (link)]. Total area for each staining was determined for complete intestinal sections and expressed as percentage of epithelial area (E-cadherin).