20 water immersion objective

Windows were prepared as previously described^{56 (link)}. Briefly, animals were anesthetized via i.p. injections of 100 mg/kg ketamine and 10 mg/kg xylazine. A 3–4 mm craniotomy was prepared over the somatosensory cortex, and the underlying dura was removed. After dye labeling, a #0 cover glass was placed over the exposed area and sealed with dental cement. For all experiments, mice were imaged whilst anesthetized with a mixture of ketamine and xylazine as stated above. For repeated in vivo imaging, a nut was affixed to the skull and embedded in dental cement for head immobilization and window orientation. Cerebral vessels were visualized by intravenous (i.v.) injection of EBD. In vivo images were acquired using an upright Leica SP5 confocal microscope. A ×20 water immersion objective (1.0 NA, Leica) was used for optimal fluorophore excitation and emission separation. For confocal imaging, the following wavelengths were used: 488 nm for GFP, 561 nm for tdTomato, and 633 nm for EBD.