Mice were anesthetized with 120 mg/kg ketamine plus 8 mg/kg xylazine (Phoenix Pharmaceuticals, St. Joseph, MO) diluted in sterile saline. Animals were placed in a stereotaxic frame with the top of the head resting 30cm below the X-ray source. A lead shield protected the body of the animals. Animals (n = 5) were exposed to cranial irradiation using a Siemens Stabilopan X-ray system operated at 300 kVp and 20 mA.

X-rays were delivered one time for 5.5 min, resulting in a dose of approximately 10 Gy. Dosimetry for this system has been reported elsewhere EGFR inhibitor (Santarelli et al., 2003). Sham-irradiated controls (n = 2) received anesthesia only. Animals were anesthetized as above and transcardially perfused with 4% paraformaldehyde (PFA). Brains were postfixed in 4% PFA overnight, cryoprotected in 30% sucrose, cryosectioned at 40 μm, and stored in PBS with 0.02% sodium azide. Free-floating sections were washed in PBS, blocked and permeablized in 10% normal donkey serum and 0.5% Triton X-100, and incubated overnight at 4°C in primary antibodies (except BLBP, 36 hr, and Nestin, 7 days) in blocking solution. For Nestin and BrdU, sections were mounted onto slides and antigen retrieval was performed. learn more The following antibodies were used: Rabbit anti-BLBP (1:1000, gift from Dr. Nathaniel

Heintz); Rat anti-BrdU (1:100, Serotec, Martinsried, Germany); Mouse anti-Calbindin (1:5000,

Swant, Bellinzona, Switzerland); Rabbit anti-Cleaved Caspase-3 (1:500, Cell Signaling Technology, Beverly, MA); Goat anti-Doublecortin (1:500, Santa Cruz Biotechnology, Santa Cruz, CA); Rabbit anti-GFAP (1:1000, DAKO, Carpinteria, CA); Chicken anti-GFP (1:500, AbCam, Cambridge, MA); Rabbit anti-GFP (1:1000, Molecular Probes, Eugene, OR); Goat anti-MCM2 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA); Mouse anti-NeuN (1:1000, Chemicon, Temulca, CA); Rabbit anti-S100β (1:5000, Swant, Bellinzona, Switzerland); Rat anti-Nestin (1:50, BD PharMingen, San Diego, CA); and Rabbit anti-Tbr2 (1:1000, AbCam, Cambridge, MA). All fluorescent PD184352 (CI-1040) secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA) and diluted 1:400 in PBS except Goat anti-Rabbit Alexa 405 (1:200, Molecular Probes, Eugene, OR). Some sections were counterstained with Hoechst 33342 (1:10,000, Molecular Probes, Eugene, OR). For quadruple labeling, sequential secondary incubation was used to avoid cross-reactivity between Goat anti-Rabbit Alexa 405 with Goat anti-Doublecortin. The Cleaved Caspase-3 antibody was visualized using ABC and a DAB kits (Vector Laboratories, Burlingame, CA). Fluorescent confocal micrographs were captured with an Olympus IX81 confocal microscope equipped with a 405 laser and the aid of Olympus Fluoview 1000v1.5 software. Representative images were edited using Adobe Photoshop.