A robust vessel-labeling pipeline with high tissue clearing compatibility for 3D mapping of vascular networks.

The combination of vessel-labeling, tissue-clearing, and light-sheet imaging techniques provides a potent tool for accurately mapping vascular networks, enabling the assessment of vascular remodeling in vascular-related disorders. However, most vascular labeling methods face challenges such as inadequate labeling efficiency or poor compatibility with current tissue clearing technology, which significantly undermines the image quality. To address this limitation, we introduce a vessel-labeling pipeline, termed Ultralabel, which relies on a specially designed dye hydrogel containing lysine-fixable dextran and gelatins for double enhancement. Ultralabel demonstrates not only excellent vessel-labeling capability but also strong compatibility with all tissue clearing methods tested, which outperforms other vessel-labeling methods. Consequently, Ultralabel enables fine mapping of vascular networks in diverse organs, as well as multi-color labeling alongside other labeling techniques. Ultralabel should provide a robust and user-friendly method for obtaining 3D vascular networks in different biomedical applications.

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.