Hippocampi were dissected from E18 embryonic rat’s brain in a 3°C pre cooled Ca2+ and Mg2+ free HBSS solution and then incubated with 0.25% of trypsin for 15 min at 37°C. Hippocampi were washed two times with a HBSS solution. Tissue was triturated by using a fire polished Pasteur pipette. The neurons and glial cells were plated at a density of 200x103 cells / mL in a 12 mm culture dishe, with 0.5 mL of culture medium per dish. Culture medium contains MEM and supplements (Invitrogen).
Cells have been transfected at 14 or 21 d.i.v (day in vitro). 0.3 µg of a GFP or a GFP-NR2B plasmid DNA was mixed with 2.5 µL of Lipofectamine™ 2000 reagent (Invitrogen) with or without 0.3 µL of CombiMag reagent (Magnetofection™ from OZ Biosciences). 1 µL of PolyMag reagent and 3 µL of NeuroMag from OZ Biosciences were also used with 1 µg of plasmid DNA.All complexes were incubated at room temperature for 20 minutes and then added drop by drop to the cells. Finally, the cell culture dishes were placed upon the magnetic plate (OZ Biosciences) for 15 minutes at 37°C. The cells were incubated at 37°C in a CO2 incubator under standard conditions until the evaluation of the plasmid expression level.
Cells were fixed with a 2 % formalin solution followed by 1 minute of incubation in a cold methanol solution (-20°C). To block non-specific antibody binding sites, 1% of a goat serum was added and neurons were incubated for 30 minutes at room temperature. Neurons were then stained with a mouse MAP-2 antibody (Abgent) and revealed with a secondary red Alexa-546 antibody (Invitrogen).