Organotypic cultures of mouse coronal forebrain slices were prepared following published methods45 with some modifications. Whole brains from E14–E18 mouse embryos were embedded in 4% low-melting point agarose and slices were cut at 250–300 μm using a Leica VT1200 vibrotome in complete HBSS (100 ml of 10× HBSS without Ca or Mg, 2.5 ml of 1M HEPES buffer at pH 7.4, 30 ml of 1M D-glucose, 10 ml of 100 mM CaCl2, 10 ml of 100 mM MgSO4, and 4 ml of 1 M NaHCO3). Slices with visible forebrain structures were placed in membrane inserts (diameter, 13 mm; pore size, 8 μm; Costar) coated with Poly-L-orthinine and Laminin (Sigma) overnight. They were cultured in a Basal Medium Eagle (39 mL, Life Technologies, #21010046) supplemented with 12.9 ml of complete HBSS, 1.35 ml of 1M D-glucose, 250 μl of 200 mM GlutaMax (Life Technologies) and 5% heat-inactivated horse serum (Life Technologies, 26050070). Slices were imaged using a Leica SP8 confocal microscope. Approval for rodent experiments was obtained from the Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC).
