Organotypic Brain Slice Culture Preparation

Organotypic brain slice cultures of 8-day-old C57BL/6 mice were prepared and maintained according to an established protocol [17 (link)]. Animal brains were removed and kept under ice-cold conditions. The cerebellum was removed. The remaining brain was embedded in 5% low melting agarose and cut into 350 µm thick coronal slices using a vibratome (Leica VT1000S, Bensheim, Germany). The brain slices were placed onto cell culture inserts (pore size 0.4 µm; Greiner BioOne, Frickenhausen, Germany) and subsequently transferred into six-well culture dishes (GreinerBioOne) containing 1.2 mL culture medium (MEM-HBSS, 2:1, 25% horse serum, 2% L-glutamine, 2.64 mg/mL glucose, 100 U/mL penicillin, 0.1 mg/mL streptomycin, 10 µg/mL insulin-transferrin-sodium selenite supplement, and 0.8 µg/mL vitamin C–pH was adjusted to 7.4 with 37% HCl and the medium was stored at 4 °C for up to 2 weeks). The slices were cultured in a humidified atmosphere (35 °C, 5% CO₂) for up to 21 days. The medium was changed on the day following OBSC preparation and thereafter every second day. At the end of the experiments, the brain slice thickness was reduced to 100 μm, as expected for an organotypic culture.