Quantifying Intestinal Cell Apoptosis

Cell apoptosis in intestinal tissue was visualized using TUNEL assay similar to described before^{48 (link)}. Briefly, ileal tissue slides were deparaffinized with xylene bath for 3 times and then rehydrated with 100%, 95%, and 70% ethanol. The tissue was then incubated with TUNEL solution (5 µM Fluorescein-12-dUTP (Enzo Life Sciences), 10 µM dATP, 1 mM pH 7.6 Tris-HCl, 0.1 mM EDTA, 1U TdT enzyme (Promega)) at 37 °C for 90 min. The slides were counter-stained with DAPI for nucleus visualization. The fluorescent green apoptosis cells were evaluated and imaged using a Nikon TS2 fluorescent microscopy. The green dots in representative 3 areas per slide were counted as apoptosis cells and blue dots of nuclei were counted as total cells using ImageJ^{49 (link)} particle analysis and its plugin of Nikon ND2 reader. The results were showed as apoptosis cells per 1,000 total intestinal cells.