Millicell insert well

E13.5 to P1 animals were perfused with 5 ml of ice-cold sucrose solution (70 mM sucrose, 86 mM NaCl, 4 mM KCl, 1 mM NaH₂PO₄, 7 mM MgCl₂, 26mM NaHCO₃, 25 mM glucose, 0.5 mM CaCl₂). Cortices were dissected and embedded in 4% low melting agarose in PBS. Upon solidification of the agar block, the brain-agarose block was glued onto a vibratome and cut in ice-cold sucrose solution at 250 μm. Slices were transferred into an ice-cold solution of KREB to recover for 15 min before being placed into a Millicell insert well (Millipore) and incubated at 37ºC in 5% CO₂ with MEM/10% FBS/1% Penstrep. After 1 h at 37ºC, slice medium was changed to Neurobasal/B27. Slices were incubated in Neurobasal/B27 for up to 1 week without any changing of medium.