Many experimental animal models of human neurodegenerative diseases have been developed to understand the events leading toward neuronal dysfunction and death. However, definitive comprehension of the molecular and cellular mechanisms in these animal models is problematic because of the complexity of the intact nervous tissue. Primary neuronal cultures prepared from rodent nervous tissues represent a powerful tool not only to study the individual contribution of different cell types (such as neurons or glia) to disease progression, but also to investigate the role of neuron-glia interactions during development and pathogenesis of disease. Here, we describe a method to isolate and culture neurons and astrocytes from the mouse cerebral cortex, and we also present a practical application for transfection and subsequent immunofluorescence.