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For Caspase-3 staining, the tissues were blocked with 10% normal horse serum for 30 min then incubated with Caspase-3 antibody (1:300, BD Biosciences, Macquarie Park, NSW, Australia) overnight, followed by secondary antibodies (Goat anti-rabbit HRP, 1:200, Vector Laboratories, Burlingame, CA, USA) for 45 min