Effective labeling of the vascular system is essential for understanding the vascular networks of tissues and organs. Here, we present VALID, a vascular labeling method that visualizes vascular networks through tissue clearing and light-sheet microscopy. VALID converts traditional lipophilic dye solutions into hydrogels by introducing gelatin and inhibits the aggregation of dyes, thus obtaining a complete and uniform vascular labeling pattern with high signal-to-value ratio. VALID enhances the compatibility of lipophilic dyes with solvent-based tissue clearance regimens, which was previously difficult to achieve. Using VALID, we combined lipophile dyes with solvent-based tissue clearing to perform 3D reconstruction of the vascular system within the brain and spinal cord of mice. We also used VALID to visualize and quantify microvascular damage in a mouse model of middle cerebral artery occlusion. VALID offers a simple, cost-effective vascular labeling protocol that will greatly expand the use of lipophilic dyes in the study of cerebrovascular complications.
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