INTRODUCTIONRodent organotypic hippocampal slice cultures (OHSCs) provide an outstanding preparation of central nervous system tissue for exploring the dynamic structural and physiological features of neuronal and glial cells within their native three-dimensional environments. It is a straightforward matter to obtain tissue slices from neonatal rodents. These slices culture well for periods up to several weeks and are easy to manipulate, allowing for a variety of in vitro experimental models. OHSCs provide good optical and physiological accessibility for studies involving live cell imaging, with high spatial and temporal resolution. This protocol is used to harvest tissues for both immunohistochemical labeling after fixation, and for confocal time-lapse imaging in live tissues labeled by a variety of fluorescent dyes or by biolistic or viral transfection.