Goat anti sox2

After culture and imaging, slices were fixed in 4% PFA at 4°C for 4 h, and washed thoroughly in PBS. Slices were blocked in 10% normal horse serum (NHS) and 0.4% Triton-X in PBS for 1 h and incubated in primary antibody (in PBS, 2.5% NHS and 0.4% Triton-X) for 48 h at 4°C. Primary antibodies included: rabbit anti-Olig2 and mouse anti-NeuN (1:500; Millipore, Germany), rat anti-Ki67 (1:500; eBiosciences, San Diego, CA), goat anti-GFAP (1:2000) and goat anti-SOX2 (1:500; Abcam, UK) and rabbit anti-GFAP (1:500; Dako, Denmark). After PBS rinses, slices were incubated in the appropriate secondary antibody conjugated to Alexafluor 405, 594, or 633 (1:500; Life Technologies, Carlsbad, CA) in PBS with 2.5% NHS and 0.4% Triton-X overnight at 4°C. Slices were washed in PBS, placed between two coverslips with a 400 um thick slice of agarose to act as a spacer if needed and mounted in Fluoromount-G (eBiosciences, San Diego, CA).
All fixed tissue was imaged with a laser-scanning confocal microscope (Olympus FV1000, Germany) equipped with a UV diode (405 nm excitation) and three laser lines (488, 543, 633 nm) along with the appropriate emission filter sets; this configuration allowed for imaging of GFP expression and up to three additional markers in a single slice (for example see Figure 4A).