2.7. TUNEL Assay to Determine Neural Cells Apoptosis

The formalin-fixed tissue was embedded in paraffin and sectioned at 4 μm thickness with a microtome. The sections were analyzed by the TUNEL assay to detect the apoptotic cells. The TUNEL assay kit was purchased from Roche Molecular Biochemicals, Mannheim, Germany, and the experiment was conducted according to manufacturer's protocol. Apoptotic cells with condensed nuclei were stained brown in the TUNEL assay, while normal cells were large, round, and not stained. The positive cells were analyzed under a light microscope by an investigator blinded to the experimental groups. The extent of brain injury was evaluated by the percentage of TUNEL-positive cells.