High-speed live imaging of ciliary beating and ependymal flow assay were performed as described previously with minor modifications41 (link),42 (link). For the high-speed imaging of ciliary beating, the wholemount preparations of the lateral walls of the LVs were incubated with FITC-labelled rat anti-CD24 antibody (BD Biosciences) in DMEM (Nacalai) for 30 min at RT, rinsed with DMEM, placed on a dissection dish, and fixed with staples. Ciliary beating was recorded with a 10 ms exposure time at 100 frames per second (fps) at RT using an Olympus BX53 microscope, LUMFLN60XW water immersion objective lens (NA 1.10), ORCA-Flash4.0 V3 high-speed camera (HAMAMATSU) and high-speed recording (HSR) software (HAMAMATSU).
For the ependymal flow assay, a glass micropipette filled with fluorescent polystyrene latex microbeads (2 µm, Polysciences) attached to an MO-10 micromanipulator (Narishige) was lowered onto the wholemount, and the microbeads were deposited onto the ventricular surface. The movement of microbeads was recorded at RT at 20 fps using an Olympus SZX16 fluorescent dissection microscope, ORCA-Flash4.0 V3 high-speed camera and HSR software. The speeds of the migrating fluorescent beads were quantified using the Manual Tracking plugin for ImageJ software (written by Dr. Fabrice P. Cordelieres).
For the ependymal flow assay, a glass micropipette filled with fluorescent polystyrene latex microbeads (2 µm, Polysciences) attached to an MO-10 micromanipulator (Narishige) was lowered onto the wholemount, and the microbeads were deposited onto the ventricular surface. The movement of microbeads was recorded at RT at 20 fps using an Olympus SZX16 fluorescent dissection microscope, ORCA-Flash4.0 V3 high-speed camera and HSR software. The speeds of the migrating fluorescent beads were quantified using the Manual Tracking plugin for ImageJ software (written by Dr. Fabrice P. Cordelieres).