Visualizing Meningeal Lymphatic Vessels

Adult Prox1^GFP, Lck^Cre::tdTOMATO^fl/fl mice were anesthetized by ketamine/xylazine intraperitoneal (i.p.) injection. Mice were injected intravenously (i.v.) or into the cisterna magna (i.c.m.) with Qtracker655 vascular tracker (ThermoFisher - Qdot⁶⁵⁵ – 5µl with 95µl saline (i.v.) or 5µl (i.c.m.)). Furamidine dihydrochloride (125µg -Sigma Aldrich) was injected i.v. prior to imaging. Meningeal lymphatic vessels were imaged through a thinned skull preparation above the superior sagittal, transverse sinuses or cerebellum. The core temperature of the mice was maintained at 37C. Imaging was performed with a Leica TCS SP8 multiphoton system (Leica Microsystems) equipped with a Chameleon Ultra II tunable Ti:sapphire laser (Coherent). GFP, tdTOMATO and Qdot⁶⁵⁵ were excited with an excitation wavelength of 880 nm (or 780nm for furamidine^{57 (link)}). Second harmonic signal (bone and meningeal signal) was visualized using a 400/450 external detector. Images were obtained using a 25X water immersion objective with 0/95NA and external HyD non-descanned detectors (Leica Microsystems). Three-dimensional images data were collected by obtaining images from the x,y and z-planes. The resulting images were analyzed with Imaris software (Bitplane).