Mouse monoclonal anti gad67 antibody

Example 2

Experiments were conducted according to National Institutes of Health guidelines for animal research and were approved by the Janelia Farm Research Campus Institutional Animal Care and Use Committee. Neonatal rat pups were sacrificed and hippocampi were dissected and dissociated in papain (Worthington, ˜10 U/hippocampal pair) in neural dissection solution (10 mM HEPES pH 7.4 in HBSS) for 25 min at 37° C. Following trituration with a Pasteur pipette and passage through 40-μm strainer, cells were plated at a density of 2.25×10⁵ viable cells/well in 150 μL plating medium (28 mM glucose, 2.4 mM NaHCO₃, 100 μg/mL transferrin, 25 μg/mL insulin, 2 mM L-glutamine, penicillin/streptomycin, 10% fetal bovine serum in MEM) in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips). Wells were pre-coated with 100 μL Matrigel (1:50 dilution in MEM, BD Biosciences), which was aspirated immediately before plating cells. After 1 h at 37° C., 1 mL plating medium was added to wells. After 16 h, plating medium was replaced with 1 mL growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 μg/mL transferrin, B-27 supplement (1×, Invitrogen), 500 μM L-glutamine, penicillin/streptomycin, 5% fetal bovine serum in MEM). A mouse monoclonal anti-GAD67 antibody (Millipore) was used to stain fixed cultures.

Example 2

Experiments were conducted according to National Institutes of Health guidelines for animal research and were approved by the Janelia Farm Research Campus Institutional Animal Care and Use Committee. Neonatal rat pups were sacrificed and hippocampi were dissected and dissociated in papain (Worthington, ˜10 U/hippocampal pair) in neural dissection solution (10 mM HEPES pH 7.4 in HBSS) for 25 min at 37° C. Following trituration with a Pasteur pipette and passage through 40-μm strainer, cells were plated at a density of 2.25×10⁵ viable cells/well in 150 μL plating medium (28 mM glucose, 2.4 mM NaHCO₃, 100 μg/mL transferrin, 25 μg/mL insulin, 2 mM L-glutamine, penicillin/streptomycin, 10% fetal bovine serum in MEM) in 24-well glass-bottom plates (Mattek, #1.5 glass coverslips). Wells were pre-coated with 100 μL Matrigel (1:50 dilution in MEM, BD Biosciences), which was aspirated immediately before plating cells. After 1 h at 37° C., 1 mL plating medium was added to wells. After 16 h, plating medium was replaced with 1 mL growth medium (28 mM glucose, 2.4 mM sodium bicarbonate, 100 μg/mL transferrin, B-27 supplement (1×, Invitrogen), 500 μM L-glutamine, penicillin/streptomycin, 5% fetal bovine serum in MEM). A mouse monoclonal anti-GAD67 antibody (Millipore) was used to stain fixed cultures.