Cultured organotypic hippocampal slices (hOTCs) have become increasingly popular as a model for studying brain function. This model offers significant advantages over traditional in vitro methods, as they allow the examination of mid to long-term manipulations while preserving the structure of the dentate gyrus (DG) in the hippocampus. In this chapter, we focus on a protocol based on hOTCs of mouse entorhinal cortex and hippocampus, which by integrating techniques such as retroviral injections, immunohistochemistry, and microscopy imaging, physiological or pathological processes can be easily investigated.