All mice were injected with 100 mg/kg BrdU (Sigma-Aldrich, St. Louis, MO) 2 hr prior to sacrifice. The intestinal tissues were harvested and processed in bundles as described (20 (link), 27 (link)) (Supplemental Material). Histological analysis was performed by hematoxylin and eosin (H&E) staining. Mitoses were scored by visual inspections of H&E sections under microscope at magnification of 600× (28 (link)). Normal mitoses contain condensed chromosomes that show even and symmetrical separation and alignment. Aberrant mitoses contain condensed chromosomes with multi-polar spindles, lagging or misaligned chromosomes, anaphase bridges, or micronuclei.
TUNEL and BrdU staining were performed as described (20 (link)) (Supplemental Material). In brief, TUNEL staining was performed with the ApopTag Peroxidase Kit or ApopTag Fluorescein In Situ Apoptosis Detection Kit (Chemicon International, Temecula, CA). Complete crypts extending to neighboring villi and containing at least 17 cells along either side with several Paneth cells at the bottom were used for counting in samples collected up to 48 h after irradiation unless indicated otherwise. TUNEL-positive or BrdU-positive cells were scored in 100 crypts/mouse, with a minimum of three mice per group. Data were reported as means ± SEM.
The crypt microcolony assay was used to quantify stem cell survival by counting regenerated crypts in H&E stained cross sections 3 and 4 days post-irradiation (20 (link)). More details are found in the Supplemental Material. At least three mice were used in each group and the data are reported as means ± SEM.