TUNEL Assay

For the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, apoptosis in paraffin-embedded sections was assessed using an In Situ Cell Death Detection Kit In (TMRM Red; Roche, Branchburg, NJ) according to the manufacturer's instructions. Slides were visualized under Nikon E600 brightfield microscope, and digital images were taken with iVision software. Images were taken to cover the entire OTC section. We analyzed three different tissue sections per OTC, and three separate OTCs for each treatment. All images were analyzed with ImageJ software (NIH), counting all of the nuclei and positive cells.