BrdU Labeling of Intestinal Epithelial Cells

Proliferating intestinal epithelial cells were stained with 5-Bromo-2’deoxyuridine (BrdU). At 90 minutes prior to sacrifice, mice received intraperitoneal injections of BrdU (5mg/ml in 0.9% saline, Sigma-Aldrich) to label S-phase cells [23 (link),24 (link)]. Jejunal tissue was then fixed in 10% formalin for 24 hours before being embedded in paraffin and slide-mounted in 5μm sections. Slides were then deparaffinized, rehydrated, and incubated in 1% hydrogen peroxide for 15 minutes before being heated in a pressure cooker in antigen decloaker (Biocare Medical, Concord, CA) for 45 minutes. Protein block (Dako, Carpinteria, CA) was performed for 30 minutes at room temperature and slides were incubated overnight at 4°C with rat monoclonal Anti-BrdU (1:500; Accurate Chemical and Scientific, Westbury, NJ). Samples were then incubated with goat anti-rat antibody (1:500; Accurate Chemical & Scientific) and streptavidin horseradish peroxidase (1:500; Dako), each for an hour at room temperature. Diaminobenzidine (DAB) was used to develop slides for 2–3 minutes, and counterstaining was performed with hematoxylin. BrdU-positive cells were quantified in 100 contiguous, well-oriented intestinal crypts.