Organotypic brain slice culture

Organotypic brain slices were obtained from neonatal mice on postnatal day (PND) 5 and cultured according to the Interface method as first described by Stoppini et al. in 1991 with some modifications [20]. Pups were rapidly decapitated, and brains were extracted and glued onto the stage of an Integraslice Microtome after cerebella were removed. The stage was submerged in a water bath filled with derivation medium (MEM +20 mM Hepes +1% P/S) that was cooled to 3°C during the whole procedure. The brains were cut into 350 μm-thick sections with a frequency of 80 Hz, and the central five slices were used. Each slice was placed on top of a Millicell Cell Culture Insert (diameter 30 mm, pore size 0.4 μm) and the insert was transferred to a six-well plate, where every well contained 1 mL of tissue culture medium consisting of 50% MEM, 25% HBSS, 25% heat-inactivated horse serum, supplemented with additional 12.5 mM Glucose, and 1% P/S.

After derivation, slices were washed twice and the six-well plates were placed in a humidified incubator with 5% CO₂ at 37°C. The medium was changed the day after derivation and then every 2 days with a gradual change to GRP medium so that from the seventh day onward all slices received GRP medium.