Neonatal Intracerebral Transplantation for Microglia Replacement and Lineage Analysis In Vivo

Microglia replacement via myeloid cell transplantation has emerged as a promising experimental approach for studying neurological processes and disease. However, existing transplantation strategies are often limited by inefficient engraftment, reliance on irradiation-based conditioning, or restricted flexibility across donor and recipient systems. Here, we describe a neonatal intracerebral transplantation protocol for introducing purified microglia populations (Hoxb8 and non-Hoxb8 microglia) into the developing mouse brain. This method enables efficient donor cell engraftment and integration within the brain parenchyma under physiologically permissive conditions. Importantly, the protocol is compatible with multiple donor sources, including embryonic hematopoietic progenitors and postnatal brain-derived microglia, and can be applied across distinct recipient models with reduced or absent endogenous microglia, including conditional Csf1r-deficient mice and Csf1r^ΔFIRE mice. Donor cells are purified by fluorescence-activated cell sorting and delivered bilaterally into neonatal mouse brains using glass micropipettes for efficient and minimally invasive delivery. Recipient mice are aged to allow donor cells to expand and populate the microglia niche, and outcomes are assessed by histological analysis of donor cell distribution and marker expression. This approach provides a robust and scalable platform for studying microglia ontogeny and in vivo integration across diverse experimental contexts.