Ex vivo hippocampal slice preparation and imaging.

Hippocampal tissue slices (400 μm thick) prepared from P14–P21 mice were incubated in imaging media. Microglia were identified by genetically induced fluorescence (Cx3cr1^gfp/+). For time series, we collected 15 Z-steps per image, spaced 2 mm apart (30 mm total depth). Images were taken up to 1 hour, at 5-minute intervals, on a Scientifica 2-photon microscope with ×20 lens. Image processing and analysis were performed using NIH ImageJ software.