Immunohistochemical analysis for caspase-3

Heart tissue sections (4 µm thick) were deparaffinized with xylene and then antigens were unmasked by immersing the sections in 0.1 M sodium citrate buffer (pH 6) in heated water bath for 15 min, followed by endogenous peroxidase blocking in 3% H₂O₂ for 10 min to block the endogenous peroxidase activity. After cooling, nonspecific binding was blocked by incubating with non-fat dried milk. The primary antibodies caspase 3 (sc-65497) was obtained from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA). The sections were incubated with primary antibodies overnight at 4 °C. After washing the slides with PBS, then sections were incubated with polyvalent biotinylated goat anti-rabbit secondary antibody diluted 1: 200 at room temperature for 10 min. After a standard staining protocol using Universal LSAB plus kit and a DAB plus substrate kit as the chromogen, sections were counter-stained with hematoxylin. Tissue images were captured by optical microscopy (Olympus DP71). Then, intensity of dark-brown staining was quantified in ten randomly selected fields (magnification 400x) per individual samples by using ImageJ software (1.39, NIH-Bethesda, MD, USA).