Caspase 3 rabbit monoclonal antibody

Apoptotic hepatocytes were identified using cleaved-caspase-3 / pro-caspase-3 immunohistochemistry. For caspase-3 immunostaining, 5μm-sections were dewaxed and hydrated through graded ethanols, cooked in 10 mM citrate buffer at pH 6.0 in a pressure cooker/antigen retriever at 125°C for 30 min (2100-Retriever, Electron Microscopy Sciences, Hatfield, PA, USA), then transferred into water to cool for 10 min. After 5 min of treatment in 3% H₂O₂, the slides were blocked with 10% normal goat serum (NGS) for 1 h. After blocking with 10% NGS, the slides were dried, and covered with the primary antibody (caspase-3 rabbit monoclonal antibody 1: 800 and pro-caspase-3 rabbit polyclonal antibodies 1:2000, Cell Signaling) overnight at 4°C. The slides were then washed three times and incubated with the secondary antibody (biotinylated goat anti-rabbit) for 1 hr. Following this, the slides were washed and dried, and then covered with ABC solution (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 h followed by the addition of DAB solution (Vector Laboratories) for 2 min. Later, DAB solution was washed and counterstained with Mayer’s Haematoxylin for 2 min followed by rinsing with tap water 2–3 times. The slides were then immersed in Scott’s solution for 2 min, and dehydrated and mounted with a coverslip.