Slice Cultures

Procedure

Organotypically cultured hippocampal slices were isolated from P5-P6 rats and prepared using the modified interface culture method described by Stoppini et al., 1991, in accordance with the European Communities Council Directive N. 86/609/EEC. Wistar pups were decapitated, and the brains were rapidly dissected and placed in icecold Dissection medium (HBSS 1X; HEPES 20 mM; Pen/Strep100 U/ml; D-glucose 6 mg/ml); the hippocampi were isolated and sectioned into 350-mm transverse slices with a McIlwain tissue chopper (Mickle Laboratory Engineering, Goose Green, UK). The slices were then carefully separated and transferred onto porous membrane inserts (four slice per insert; Millicell-CM, Millipore) of 6-well culture
plates. 1200 lL culture medium, consisting of NeurobasalA medium, supplemented with 13 B27 (from Invitrogen), 1 mM Lglutamine, 100 U/mL penicillin, and 100 lg/mL streptomycin, 25% heat inactivated horse serum (Hyclone) were added to the lower compartment of each well and the culture plates were then placed in a 35 C humidified incubator enriched with 5% CO2. On the next day of culture, the culture medium was replaced with fresh medium and, from that time, changed twice a week.

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