This protocol details a method to establish organotypic slice cultures from mouse hippocampus, which can be maintained for several months. 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 – from when they are isolated at postnatal day 6–9, and up to 6 months in vitro. The preserved tissue architecture facilitates the analysis of defined hippocampal synapses, cells and entire projections. Monitoring of defined cellular and molecular components in the slices can be achieved by preparing slices from transgenic mice or by introducing transgenes through transfection or viral vectors. This protocol can be completed in 3 h.