Brains were collected from two to three P8 animals, kept in iced HBSS with bubbled oxygen, and cut into 250-μm sections with a Leica VT1000 S vibratome. Sections containing lateral ventricle were incubated in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) at 37°C for 5 min with membrane-permeable dyes: Hoechst nuclear stain (5 μg/ml) (H3570, Invitrogen), Lectin DyLight 488 (5 μg/ml) (DL-1174, Vector Laboratories) for ependymal and vascular staining, and Vybrant CM-DiI (5 μg/ml) (V22888, Invitrogen). CM-DiI was used to nonspecifically label cell membranes; however, ependymal cilia had higher staining intensities than other cellular membranes, likely because of their surface area and direct exposure to solution. Following two 3-min washes in DMEM, brains were transferred to a glass-bottomed 12-well plate (P12G-1.5-14-F, MatTek) containing 1 ml of high-glucose DMEM and weighed down with harp slice grids (HSG-5BD MEA, ALA Scientific). Sections were incubated for 1 hour to induce adhesion before imaging on a Nikon N-SIM with an A1R confocal scanner and equipped with a plate-heating chamber. This microscope has a resonance scanner that acquires simultaneous images at 405-, 488-, and 568-nm wavelengths. By performing a band scan, we were able to capture 10-s time-lapse images at 113.2 fps, which allows for visualization of ciliary beating. These videos were analyzed with Imaris to track the movement of CM-DiI–stained cilia. Points were excluded for colocalization with Hoechst or Lectin 488, restricted by frame-to-frame distance to track the back-and-forth movement of cilia, and filtered by tracking duration to prevent quantification of cilia beating out of frame. Tracking analysis video was exported at 23 fps (1/5 speed).

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