Rabbit monoclonal antibody against cleaved caspase 3

To process brain tissue for histological and immunohistochemical examination, mice were anesthetized with 5% isoflurane. The mice were then transcardially perfused with heparinized saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4). After postfixation for several hours, the brains were removed and embedded in paraffin, and cut into 6 µm-thick coronal sections. Slides were stained with cresyl violet according to the Nissl method and evaluated for evidence of cellular degeneration. Cells that contained Nissl substance in the cytoplasm, loose chromatin, and prominent nucleoli were considered to be normal neurons, and ischemic neurons were identified by loss of Nissl substance and by the presence of pyknotic homogenous nuclei. Activation of caspase-3 was assessed by immunohistochemistry in paraffin-embedded coronal sections using a rabbit monoclonal antibody against cleaved caspase-3 (1:50, Cell Signaling) according to the protocol recommended by the manufacturer. For the quantitative analysis, the number of activated cleaved caspase-3 positive neuron in hippocampal CA1 region under high-power magnification (200x) by an investigator (TK) blinded as to the identify of mice.