In Situ Imaging of Cerebral Vasculature

The method for in situ imaging was established in the Zipfel group (Aum et al., 2017 (link)). Mice were anaesthetized using a ketamine (Bayer, Reading, UK) and xylazine (Pfizer, Tadworth, Surrey, UK) mixture (100 and 10 mg/kg, respectively) and transcardially perfused through the left ventricle with 10 mM glucose–physiological-buffered saline (PBS) followed by 20 ml 20 μM 5-(6)-carboxy-X-rhodamine, succinimidyl ester (Sigma-Aldrich, Gillingham, Dorset, UK) dye in 10 mM glucose-PBS prior to perfusions with 4% paraformaldehyde (in PBS). All perfusions were performed with solutions at 21°C, at a constant pressure of 80 ± 2 mmHg using a GE Druck DP1705. Animals were decapitated, the calvaria removed and post-fixed in 4% paraformaldehyde in the dark at 4°C for 24 h. Brains were removed under a dissection microscope to preserve the basal arteries. Then, brains were placed en bloc on a glass coverslip and gently covered in PBS before placing on the stage of a confocal laser scanning microscope (SP8, Leica, Wetzlar, Germany). Measurements of anterior and middle cerebral artery (MCA) diameters were made at the narrowest point across the first millimetre of the vessel, as detailed in Supplementary material.