Vp k451

This procedure was performed as described previously (19 (link)). Where indicated, immunostaining was preceded with in situ cell fractionation to reveal insoluble proteins (24 (link)). Fluorescent signals were imaged with a Zeiss CLSM710 using a 63× oil (NA = 1.4) objective. Repair foci were quantified using an automated routine in ImageJ (http://rsbweb.nih.gov/ij/) or by visual scoring. Antibodies used for immunostaining were: 53BP1 (Abcam, ab36823, 5 μg/ml), BRCA1 (Calbiochem, MS110, 0.3 μg/ml), cyclin B1 (Cell Signaling Technology, clone D5C10, 1:200), FLAG (Sigma, M2, 2 μg/ml), γH2AX (Milipore, JBW301, 3.3 μg/ml), Histone H4 acetylated (Milipore, 06–598, 5 μg/ml), Histone H4K20me (Abcam, ab9051, 5 μg/ml), Ki67 (Vector Laboratories, VP-K451, 1:1000), lamin B (Abcam, Ab16048, 2 μg/ml), NuMA (B1C11, 1:2), NuMA full-length (Abcam, ab36999, 1:100), NuMA proximal coiled-coil (Bethyl Laboratories, A301–510A, 4 μg/ml), NuMA distal C-terminal domain (Abcam, clone EP3976, 1:250), RAD51 (Abcam, ab63801, 1:1000), SNF2h (Abcam, ab3749, 15 μg/ml or clone 3.25(2), 4 μg/ml) and WSTF (Abcam, clone EP1704Y, 1:100). Note that for immunostaining with RAD51 antibodies, a permeabilization step with 0.5% triton X100 was performed after fixation with paraformaldehyde.

To determine the differentiation and malignancy of GBM tumor in vivo, 0.3 × 10⁶ human GBM 276 BTICs were stereotactically injected into the right striatum (AP=−0.8 mm, H=3.0 mm) of immunosuppressed nude mice. Thirty days post injection, 0.5 × 10⁶ GFP/luciferase-hAMSCs or PBS were stereotactically injected into the brain (AP=−0.8 mm, H=1.5 mm) of the same side. After 3 weeks, the mice were perfused. The perfused mice brain sections were immunostained for stemness marker Nestin (Millipore, MAB5326), astrocytic marker GFAP (DAKO Z0334, Glostrup, Denmark), neural marker Tuj1 (Covance, Princeton, NJ, USA; PRB-435p), proliferation marker Ki-67 (Leica VP-K451), necrosis marker TNF-α (Abcam, ab6671), and pro-angiogenic marker VEGF (Abcam ab46154). GBM cells were identified from the background of mouse brain cells by staining for human nuclei (red) and DAPI (blue). Although injected GFP/luciferase-hAMSCs did express GFP, the green signal from GFP fluorescence was significantly weaker than the green signal of immunostaining for Nestin, GFAP, and Tuj1, and therefore did not interfere with the identification of these green fluorescence-marked proteins. Quantification of cells was performed.