This protocol details a method for imaging organotypic slice cultures from the mouse hippocampus. The cultures are based on the interface method, which does not require special equipment, is easy to execute, and yields slice cultures that can be imaged repeatedly after they are isolated on postnatal day 6-9 and for up to 6 months in vitro. The preserved tissue architecture facilitates the analysis of defined hippocampal synapses, cells and entire projections. Time-lapse imaging is based on transgenes expressed in the mice, or on constructs introduced through transfection or viral vectors; it can reveal processes that develop over time periods ranging from seconds to months. Imaging can be repeated at least eight times without detectable morphological damage to neurons. Subsequent to imaging, the slices can be processed for immunocytochemistry or electron microscopy, to collect further information about the structures that have been imaged. This protocol can be completed in 35 min.