Protocol for co-culture of primary neurons and organotypic hippocampal brain slices from mice

Here, we present a protocol for establishing a co-culture system combining organotypic hippocampal slices with primary mouse neurons to model neuronal interactions. We describe steps for preparing hippocampal slices by whole-brain dissection, tissue isolation, and sectioning. We then detail procedures for plating and culturing on membrane inserts, isolating and culturing primary neurons, and integrating to form the co-culture. The system is compatible with transgenic mice or genetically modified neurons, enabling investigation of molecular mechanisms and therapeutic targets. For complete details on the use and execution of this protocol, please refer to Zhou et al.¹.