5-ml tubes for a 30-min incubation on ice. Enteroid fragments were centrifuged at 200 g (1 min), and supernatant containing Matrigel www.selleckchem.com/products/epz-5676.html was aspirated. The enteroids were washed once with ice-cold PBS (~1 ml), centrifuged, and resuspended in PBS for plating. Proliferation and immunofluorescence. Proliferation of crypt epithelium in vivo and enteroids was measured using the Click-iT 5-ethynyl-2��-deoxyuridine (EdU) assay (Invitrogen) to label cells in S phase of the cell cycle, according to the manufacturer’s protocol. Mice were injected with EdU (4.8 ��g/g body wt) 1 h before euthanasia for the collection of duodenum. Duodenum was rinsed in ice-cold PBS, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 5-��m sections. Fluorescence (AlexFluor 488) photomicrographs of crypt cross sections were counted for EdU+ cells.

For enteroids, 4-day-old cultures (passages 1 and 2) were exposed in situ to EdU for 15 min and fixed in 4% paraformaldehyde (4��C, overnight). Fixed enteroids and Matrigel were scraped from the culture dishes, placed in 1.5-ml tubes, and centrifuged at 200 g (1 min), and supernatant containing Matrigel was aspirated. Enteroids were washed twice in PBS (~1 ml) in the same manner before the Click-iT assay was performed. Labeled enteroids were concentrated by brief centrifugation (200 g) and sealed under glass coverslips on microscope slides. Crypts were imaged on a TCS SP5 Confocal-Multiphoton microscope built on a DMI6000 inverted platform (Leica, Wetzler, Germany).

Z stacks were used to determine crypt cross sections for counting of EdU-labeled nuclei, avoiding sections where crypt fission was observed. For immunofluorescence of freshly isolated crypts, the crypts were fixed overnight in 4% paraformaldehyde, washed in PBS, and permeabilized using PBS + 0.5% Triton X-100. After blocking with PBS + 10% goat serum (30 min), the crypts were exposed overnight (4��C) to ��-catenin antibody (sc-7199; Santa Cruz Biotechnology, Santa Cruz, CA) diluted in PBS + 0.1% Triton X-100. Crypts were washed three times in PBS + 2% goat serum before incubating overnight with anti-rabbit IgG Texas Red (sc-2780; Santa Cruz; 1:100 dilution). Washed crypts (PBS + 2% goat serum) were incubated with Hoechst 33342 diluted 1:2,000 for 1 h. Crypts were suspended in buffered Fluorogel (Electron Microscopy Sciences, Hatfield, PA), mounted under glass coverslips, and imaged on the TCS SP5 Confocal-Multiphoton microscope (Leica).

Immunoblot analysis. Freshly isolated crypts and enteroids isolated by the method used for passaging (see Enteroid culture) were suspended Cilengitide in ice-cold PBS and lysed at 4��C by supersonication. Whole small intestinal epithelium from WT and Cftr KO mice was isolated by using the EDTA chelation technique, as describe previously (21), and processed similarly. Total lysate protein was loaded on 10% SDS-PAGE gels for electrophoresis, membrane transfer, and immunoblotting.