VALID achieves complete and uniform labeling of fine vascular structures in rodent brains and various organs at high signal-to-background ratios, as well as co-labeling with multiple kinds of antibodies through immunolabeling. VALID transformed traditional lipophilic dye solution into hydrogel by introducing gelatin, successfully preventing the aggregation of the dye molecules for an extended period and dye leakage during perfusion. In this study, we successfully tackle these two major challenges by developing a versatile vessel-casting method based on hydrogel-based lipophilic dye solution, termed vessel labeling via gelatin-based lipophilic dye solution (VALID). VALID should provide a simple, cost-effective vessel-labeling protocol that would significantly widen the applications of lipophilic dyes in research on cerebrovascular complications. We also employed VALID for 3D visualization and quantification of microvascular damage in a middle cerebral artery occlusion mouse model. Using VALID, we combined lipophilic dyes with solvent-based tissue-clearing techniques to perform 3D reconstructions of vasculature within mouse brain and spinal cord. VALID also enhances the compatibility of lipophilic dyes with solvent-based tissue-clearing protocols, which was hard to achieve previously.

VALID transforms traditional lipophilic dye solution into hydrogel by introducing gelatin and restrains the dye aggregation, resulting in complete and uniform vessel-labeling patterns with high signal-to-background ratios.

Here, we propose VALID, a vessel-labeling method that enables visualization of vascular networks with tissue clearing and light-sheet microscopy. Efficient labeling of the vasculature is important for understanding the organization of vascular networks.

Download mendeley word plugin for mac