Simple methodology to visualize whole-brain microvasculature in three dimensions

Significance: To explore brain architecture and pathology, a consistent reliable methodology to visualize the three-dimensional cerebral microvasculature is beneficial. Perfusion-based vascular labeling quick easily deliverable. However, quality of can vary with perfusion-based labels due aggregate formation, leakage, rapid photobleaching, incomplete perfusion. Aim: We describe simple, two-day protocol combining clearing step that facilitates whole-brain, microvascular imaging characterization. Approach: The combination retro-orbital injection Lectin-Dylight-649 label vasculature, process modified iDISCO+ protocol, light-sheet collectively enables comprehensive view cerebrovasculature. Results: observed ?threefold increase in contrast-to-background ratio over endogenous green fluorescent protein fluorescence from transgenic mouse model. With microscopy, we demonstrate sharp visualization throughout intact brain. Conclusions: Our tissue preparation requires fairly routine processing steps compatible multiple types optical microscopy.