Af3075

Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were blocked with 10% donkey serum in PBS for at least 1 h. Primary antibodies against Nestin (Abcam, ab92391), SOX2 (Abcam, ab59776), SOX1 (R&D Systems, AF3369), PAX6 (BioLegend, PRB-278P), Ki67 (CST, 9449S), Dlx2 (Abcam, ab117546), FOXG1 (Abcam, ab18259), MAP2 (Sigma Aldrich M9942), TBR1 (Abcam ab31940), BRN2 (Cell Signaling 12,137), NeuN (Abcam ab104225), SOX9 (R&D Systems AF3075), GLT1 (Novus Biologicals NBP1–20136), and GFAP (Dako Z0334) were diluted in blocking buffer and incubated overnight at 4 °C. Following several washes, the cells were incubated with the appropriate donkey anti-rabbit IgG, anti-mouse IgG, or anti-goat conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 secondary antibodies (Life Technologies) for 1 h at room temperature. Cells were counterstained with DAPI and visualized with the Leica DM6000 inverted microscope. Images were acquired using the Q-Imaging Retiga Xi Firewire High-Speed, 12-bit cooled CCD camera and Volocity software. Staining was quantified using ImageJ software and represented as a portion of total nuclei. On average, 2.5 × 10³ cells were quantified from each experiment. Results are shown with the standard deviation of the mean of three independent experiments.

In preparation for immunocytochemistry, cells were fixed in 4% PFA for 15 min at room temperature and then permeabilised for 1 hr with 0.1% Triton X-100 in PBS. After blocking with 3% BSA, samples were incubated with primary antibodies for 1 h at room temperature or overnight at 4 °C, followed by reaction with fluorescent secondary antibodies for another hour. Antibodies used were rabbit polyclonal anti-active caspase 3 (1:500, Abcam, ab2302), rabbit anti-FGF5 (1:50, Proteintech, 18171-1-AP), goat polyclonal anti-GATA4 (1:200, SantaCruz, sc-1237), chicken polyclonal anti-GFP (1:500, Abcam, ab13970), rabbit polyclonal anti-HSD3B (1:500, gift from Ian. Mason), rat polyclonal anti-Ki67 (1:500, BioLegend, 652401), rabbit polyclonal anti-laminin (1:1000, gift from Harold Erickson), rabbit polyclonal anti-MVH (1:300, Novus Biologicals, NBP2-24558), rabbit polyclonal anti-OSR1 (1:200, Abcam, ab230627), anti-Pax2 (1:200, Novus Biologicals NPB2-57700), goat polyclonal anti-SOX2 (1:200, Novus Biologicals, AF2018), goat anti-SOX9 (1:200, Novus Biologicals, AF3075), rabbit polyclonal anti-SF1/NR5A1 (1:200, Novus Biologicals NPB1-52823), rabbit polyclonal anti-STAR (1:200, Biorbyt, orb7014), rabbit polyclonal anti-STRA8 (1:200, Abcam, ab49602) and rabbit anti-SYCP3 (1:200, Novus Biologicals, NB300-230).