Western blots for Procaspase 3 and cleaved Caspase 3 were performed on tissue samples from the hippocampus of selected dogs that underwent 2-hour HCA. A block of fresh-frozen hippocampus was cut on the cryostat at 30 to 50 μm; sequential sections were collected into a series of Eppendorf tubes so that each aliquot contained a sample that extended throughout the hippocampus. Western blots were performed as previously described (16 (link), 17 (link)). Samples were sonicated in lysis RIPA buffer; a protein assay was performed and 40 μg of protein was loaded for each blot. Blots were exposed to a 1:5000 concentration of rabbit anti-ProCaspase3 (Cell Signaling Technology, Danvers, MA, #9662S) overnight at 4°C, and enhanced chemiluminescence detection (GE/Amersham) was used to visualize the bands. Two different exposure times were used to reveal Procaspase 3 and cleaved Caspase 3 (activated Caspase 3) bands on the blots. Actin served as a loading control. Images of the blots and a calibrated optical density step tablet were taken with a Microtek Scan Maker 8700 using Scan Wizardpro software; the densities of the bands were measured using MCID™ Core analysis software. Density values were analyzed from normal untreated dogs (n = 7), and at 2-hour (n = 8), 8-hour (n = 8) and 24-hour (n = 7) survival times after HCA.