In situ immunodetection of activated caspase-3 in apoptotic neurons in thedeveloping nervous system

Activation of caspase-3 requires proteolytic processing of the inactive zym ogen into p18 and p12 subunits. We generated a rabbit polyclonal antiserum, CMI, which recognizes the p18 subunit of cleaved caspase-3 but not the zym ogen. CM1 demonstrated an apparent specificity for activated caspase-3 by s pecifically immunolabeling only apoptotic but not necrotic cortical neurons in vitro. In the embryonic mouse nervous system, CM1 immunoreactivity was detected in neurons undergoing programmed cell death and was markedly incre ased in Bcl-x(L)-deficient embryos and decreased in Bax-deficient embryos. CM1 immunoreactivity was absent in the nervous system of caspase-3-deficien t mouse embryos and in neurons cultured from caspase-3-deficient mice. Alon g with neuronal somata, extensive neuritic staining was seen in apoptotic n eurons. These studies indicate that caspase-3 is activated during apoptosis in the developing nervous system in vivo and that CM1 is a useful reagent for its in situ detection.