Protocol for evaluating microglial phagocytosis in situ in mouse hippocampal slices

Microglia are the primary phagocytes of the central nervous system (CNS). Here, we present a protocol for microinjecting opsonized particles into mouse hippocampal slices followed by two-photon time-lapse microscopy. We describe steps for preparing components, removing the brain, creating coronal slices, and incubating samples. We then detail procedures for analyzing microglial morphology and cell process dynamics with microinjected particles. We also provide several common potential problems encountered during the protocol alongside troubleshooting strategies to counter them. For complete details on the use and execution of this protocol, please refer to Paulson et al.