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Paraffin-embedded 5-µm thick coronal brain sections were dewaxed, and the antigen retrieval procedure was performed. Then, the slides were incubated with a mixture of TdT and biotin-dUTP reagent provided in the TUNEL assay kit (Roche, USA) according to the manufacturer's protocol, followed by staining with streptavidin-HRP and DAB substrate. TUNEL-positive cells were counted in three non-overlapping visual fields of the ischemic cortex region selected around the infarct core for each section under a 400× light microscope (Olympus, Japan).
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