Preparation for in vivo imaging

Thirty- to sixty-day-old mice were anesthetized with isoflurane. We used 5% isoflurane for induction for up to 1 min in a chamber until the mouse was still, and 1.5%–2% for surgery for 15–30 min in the stereotactic frame. Under anesthesia, the mouse head was secured with ear bars on a heating pad and in a stereotactic frame, and a thin skull window was made with a high-speed dental drill. A head plate was glued to the skull around the cranial window, and the plate was screwed into a customized stage and placed under the two-photon microscope. Mice were maintained under light anesthesia (1% isoflurane) on the imaging stage for the duration of imaging and killed immediately after.