Ab31940

Satb2: Abcam, Mouse, ab51502, Paraffin: 1/150, Vibratome: 1/40.
Tbr1: Abcam, Rabbit, ab31940, Paraffin: 1/150, Vibratome: 1/100.
Pax6: Covance, Rabbit, PRB-278P, 1/300.
Tbr2: Invitrogen, Rat, 14–4875-82, 1/100.
Cleaved Caspase 3: Cell signaling, Rabbit, 9661, 1/300.
Eph B2: R & D Systems, Goat, AF467, WB: 1/1000.
Eph A4: Cell signaling, Rabbit, 8793, WB: 1/2000.
P-Eph B1–2: Abcam, Rabbit, ab61791, WB: 1/1000.
P-Eph A4: Gift from Greenberg’s lab, Harvard medical school, Rabbit, WB: 1/1000.

Cells were fixed in 5% PFA for 10 min at room temperature or in cold methanol at −20°C for 10 min, and blocked for 1 h in 5% donkey serum before overnight incubation in primary antibody. Primary antibodies used: Tbr1 (Abcam, ab31940; 1:500), Tuj1 (Covance, MMS-435P; 1:2000), GFAP (Abcam, ab4674; 1:1000), Otx1/2 (Millipore, AB9566; 1:200), phospho-H3 (Abcam, ab10543; 1:1000), GFP (Abcam, ab13970; 1:1000), TagRFP (Evrogen, AB233; 1:1000). Secondary antibodies conjugated with Alexa Fluor 405, 488, 546, 647 (Life Technologies) were applied after removal of primary antibody and incubated at room temperature for 1 h. Nuclei were stained with DAPI (Sigma). Confocal microscopy was performed on Leica Sp5, Olympus FV1000 Upright or Olympus-19-FV1000 Inverted microscopes. Two-photon microscopy was performed on an upright LaVision TriM Scope II multiphoton microscope equipped with a 20× objective (XLUMPlanFl 20×, NA=0.95; Olympus). Image stacks (505×505 pixels, pixel size 0.792×0.792) were acquired every 1 mm along the z-axis. Images were analysed and processed in Volocity (PerkinElmer) and ImageJ (NIH).