Protocol for Mouse Embryonic Aorta-Gonad-Mesonephros (AGM) Region Frozen Sectioning and Immunofluorescence

The aorta-gonad-mesonephros (AGM) region is the site where hematopoietic stem cells (HSCs) first emerge during development, and is therefore widely used to study in vivo hematopoiesis and to discover novel regulatory mechanisms. The endothelial-to-hematopoietic transition (EHT) process can be directly observed via immunofluorescence on frozen sections of the AGM region. However, the mouse AGM region is extremely delicate and lies deep within the embryo, between the notochord and the somatic mesoderm. Here, we present a step-by-step protocol covering embryo collection, fixation, dehydration, and embedding with a defined orientation, followed by frozen sectioning, immunofluorescence staining, and confocal imaging. The protocol is highly reproducible and easy to follow and provides clear instructions on orienting the embryo and anatomically locating AGM. By filling a technical gap, the protocol can enable researchers to reliably study HSC emergence and EHT in the mouse embryonic AGM. Key features • Provides detailed instructions on embryo embedding orientation and microscopic observation during cryosectioning to precisely locate the mouse AGM region. • Covers all steps from embryo harvesting, cryosectioning, and immunofluorescence to confocal imaging, ensuring high reproducibility. • Developed for delicate mouse embryonic AGM tissue (e.g., E9.5-E11.5), enabling direct observation of EHT via immunofluorescence.