2006; Takenaga et al. 2009). Rats on day 7 or 28 after the embolism were sacrificed by decapitation, and their whole ipsilateral Palbociclib hemisphere was homogenized in ice-cold 15 mmol/L 2-(4-2[-hydroxyethyl]-1-pioperazinyl)-ethanesulphonic acid (HEPES), pH 7.4, containing 147 mmol/L NaCl, 4 mmol/L KCl, 3 mmol/L CaCl2, and 1.2 mmol/L MgCl2 (physiological buffer). The homogenate was centrifuged at 3500g for 10 min at 4°C, and Inhibitors,research,lifescience,medical the resulting pellet was resuspended in physiological buffer containing 20% Ficoll T-400 (Sigma) and then homogenized. After centrifugation at 25,000g for 10 min at 4°C,

the pellet was resuspended in 15% dextran T-500 (Sigma). The suspension was then layered onto 20% dextran T-500 Inhibitors,research,lifescience,medical and centrifuged at 25,000g for 10 min at 4°C. The pellet was finally resuspended in physiological buffer and used as the brain capillaries. Immunoblotting Western blotting was performed according to standard protocols. The following primary antibodies were used: rabbit polyclonal antibodies against Ang-1 (Abcam, Minneapolis, MN), Ang-2 (Abcam), Occludin (Life Technologies), ZO-1 (Zymed), Tie2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),

VEGF (R&D Systems, Inc., McKinley Place, MN), and VEGFR2 (Abcam). Subsequently, the membrane was washed and Inhibitors,research,lifescience,medical incubated with secondary antibody. Bound antibody was detected by use of the enhanced chemiluminescence method (Amersham). Quantification was carried out by performing computerized densitometry with an image analyzer (ATTO Co., Tokyo, Japan). To minimize blot variability, we applied an aliquot of pooled “control” homogenate, which was obtained from naïve control rats, to one Inhibitors,research,lifescience,medical lane

of every gel and calculated the band intensity of immunoblotted samples relative to this standard. Statistical analysis The results were expressed as the means ± standard error of the mean (SEM). Differences between two groups were evaluated statistically by use of the unpaired Student’s t-test. Statistical comparison among Inhibitors,research,lifescience,medical multiple groups was made by performing analysis of variance, followed by Scheffe’s test as a post hoc test or repeated-measures Cilengitide analysis of variance. P-values of less than 0.05 were considered significant. Results Characterization of neural progenitor cells Figure 1A shows that cells in the neurospheres expressed the neural progenitor marker musashi-1 on day 6 when cultured in vitro. After triggering in vitro differentiation by withdrawal of the growth factors, we confirmed the tripotent nature of the NPCs by their ability to generate differentiated cells expressing neuronal (MAP2), astrocytic (GFAP), and oligodendrocytic (RIP) markers (Fig. 1B). Figure 1 Characterization of neural progenitor cells. (A) Triple staining with green-fluorescent protein (GFP), musashi-1, and 4′,6-diamidino-2-phenylindole (DAPI) was merged and Nilotinib CAS indicated that cells in neurospheres, which were prepared from gestational …