Para-uterine imaging protocol: Stabilizing living mouse embryos within the maternal abdomen for in vivo imaging and visually guided physiology

In vivo imaging is critical for interrogating brain circuits but is limited before birth. Here, we describe a protocol for para-uterine stabilization of living mouse embryos, enabling recordings from individual neurons and neuronal processes. We describe steps to extract embryos from the amniotic sac, place them into externally stabilized holders, expose their brains, and then transfer them for imaging, all while keeping the umbilical cords intact. Through this method, para-uterine imaging enables tools from adult head-fixed mice to generalize to embryos. For complete details on the use and execution of this protocol, please refer to Munz et al. 1 • Protocol to extract the embryo with the intact umbilical cord to maintain healthy blood flow • Steps to stabilize the embryo within the abdomen of the dam for in vivo experimental access • Instructions to obtain optical access to embryonic brain at single-neurite resolution • Guidance on the analysis of spontaneous embryonic 2p calcium imaging recordings Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. In vivo imaging is critical for interrogating brain circuits but is limited before birth. Here, we describe a protocol for para-uterine stabilization of living mouse embryos, enabling recordings from individual neurons and neuronal processes. We describe steps to extract embryos from the amniotic sac and place them into externally stabilized holders before exposing their brains and transferring them for imaging, all while keeping the umbilical cords intact. Through this method, para-uterine imaging enables tools from adult head-fixed mice to generalize to embryos.