Protocol for fine casting, imaging, and analysis of murine vascular networks with VALID

The majority of fluorescent vessel labeling techniques currently available are limited by their expense, incomplete labeling, or complexity. Here, we present VALID (vessel labeling via gelatin-based lipophilic dye solution)—a protocol for complete labeling of different vascular networks. We describe steps for preparing different dye hydrogels, murine vascular casting and tissue harvesting, immunolabeling, tissue clearing, and imaging, as well as detailed analysis of the vascular networks. This protocol is helpful for evaluating vascular lesions in studying different vessel-associated diseases. For complete details on the use and execution of this protocol, please refer to Zhu et al.1 [Display omitted] •A vessel casting protocol using lipophilic dye hydrogel•Effective for labeling complete vascular networks within different organs•Applied along with 3D immunolabeling and tissue clearing•Supports 3D visualization and analysis of different vasculature Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The majority of fluorescent vessel labeling techniques currently available are limited by their expense, incomplete labeling, or complexity. Here, we present VALID (Vessel lAbeling via geLatin-based lIpophilic Dye solution)—a protocol for complete labeling of different vascular networks. We describe steps for preparing different dye hydrogels, murine vascular casting and tissue harvesting, immunolabeling, tissue clearing, and imaging, as well as detailed analysis of the vascular networks. This protocol is helpful for evaluating vascular lesions in studying different vessel-associated diseases.