Organotypic cultures of mouse coronal forebrain slices were prepared
following published methods45 (link)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).
following published methods45 (link)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).