Ab3080

NPCs cultured on coverslips were fixed with 4% paraformaldehyde (Sigma) for 30 min and washed with phosphate-buffered saline (PBS). For GABA antibody reactivity, the cells were fixed with 4% paraformaldehyde, 0~0.5% glutaraldehyde, and 0.5% potassium dichromate in 0.1 M phosphate buffer at pH 6.5. After fixing, the cells were incubated with primary antibodies against GFP (rabbit anti-GFP antibody, 1:500, Cat. No. AB3080, Merck KGaA, Darmstadt, Germany), gamma-aminobutyric acid (mouse anti-GABA antibody, 1:1000, Cat. No. ab86186, Abcam), glutamic acid dehydrogenase 65/67 (rabbit anti-GAD65/67 antibody, 1:500, Cat. No. Ab1511, Chemicon International Inc.), and vesicular glutamate transporter 1 (mouse anti-vGluT1 antibody, 1:500, Cat. No. MAB5502, Chemicon International Inc.). Cells were subsequently incubated with FITC- or Alexa fluor-conjugated secondary antibodies (1:200 for each, Molecular Probes). Slides were counterstained with 4,6-di-amidine-2-phenylindole dihydrochloride (DAPI, 1 µg/ml, Cat. No. D9542, Sigma-Aldrich). At least five fields of view (objective magnification: ×200) from an average of five slides per experimental group were selected randomly and neuronal differentiation marker-positive cells were then counted. Stained slides were examined under a confocal laser scanning biological microscope (LSM 510, Carl Zeiss).