Immunocytochemical techniques for studying apoptosis in primary sympathetic neurons

Developing sympathetic neurons, which depend on nerve growth factor for survival, are one of the best studied in vitro models of neuronal apoptosis and have been extensively used for cellular and molecular studies of the neuronal death pathway. Important apoptotic events after nerve growth factor withdrawal include the release of proapoptotic proteins, such as cytochrome c, from the mitochondria and the activation of caspases, followed by nuclear DNA fragmentation and chromatin condensation. In this chapter, we describe immunocytochemical techniques for studying apoptotic DNA fragmentation, changes in nuclear morphology, and mitochondrial cytochrome c release at the single cell level using sympathetic neurons cultured on glass coverslips.