TUNEL identifies apoptotic cells by end-labeling the fragmented DNA present within apoptotic cells. TUNEL staining followed the manufacturer’s directions (Colormetric Kit PR-G7130 Promega, Madison, WI). Five sections per brain matched for level across the hippocampus were mounted onto Superfrost Plus® slides (Fisher Scientific) plus a positive control. Briefly, slides were washed in 0.85% NaCl then 0.1M PBS, permeabilized in Proteinase K, washed, equilibrated in equilibration buffer, and labeled by incubating in recombinant terminal deoxynucleotidyl transferase reaction mix for 1 hr at 37°C in a humidified chamber. Slides were immersed in standard sodium citrate, washed, and blocked in 0.3% H2O2. Following washes, sections were incubated in Streptavidin horseradish peroxidase, washed and colorized with DAB. Slides were dried then coverslipped with Cytoseal®.
