Protocol for live-imaging of blood flow and vascular permeability in zebrafish larvae

Cerebral vascular dynamics are critical to maintain brain homeostasis, and the fenestrated capillaries of the hypophysis (also known as the pituitary) exhibit unique structural and functional features that support brain-to-periphery neuroendocrine communication. Here, we present a protocol for live imaging of the dynamics of blood flow velocity, capillary diameter, and permeability of the hypophyseal vasculature in awake zebrafish larvae. We describe steps for two-photon microscopy-based data acquisition and the subsequent procedure for quantitative analysis of these vascular parameters. For complete details on the use and execution of this protocol, please refer to Rajamannar et al.