Visualizing brain angiogenesis in GBM

Human GBM and normal brain tissue samples were snap frozen. Intracranial xenografts were fixed in 4% paraformaldehyde, incubated overnight in 30% sucrose and embedded in OCT. Samples were sectioned at a thickness of 10µm. Blood vessels were visualized by Von Willebrand Factor (Abcam) staining. Sections were additionally stained for CD36 (Novus), integrin α6 (Millipore), CD133 (Miltenyi), and/or Iba1 (Wako). Age-matched wild-type and CD36 knockout mouse brains were prepared in a similar manner as described above and stained with antibodies against proliferating cell nuclear antigen (Abcam) and phospho histone H3 (Millipore). Analysis between wild-type and CD36 knockout mice was done based on 3 different anatomical regions from 3 separate mice. For all immunofluorescence analysis, nuclei were counterstained using 4',6-diamidino-2-phenylindole (Dapi) and images were taken using a Leica SP-5 confocal microscope as previously described [27 (link)].

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Hale J.S., Otvos B., Sinyuk M., Alvarado A.G., Hitomi M., Stoltz K., Wu Q., Flavahan W., Levison B., Johansen M.L., Schmitt D., Neltner J.M., Huang P., Ren B., Sloan A.E., Silverstein R.L., Gladson C.L., DiDonato J.A., Brown J.M., McIntyre T., Hazen S.L., Horbinski C., Rich J.N, & Lathia J.D. (2014). Cancer stem cell-specific scavenger receptor CD36 drives glioblastoma progression. Stem cells (Dayton, Ohio), 32(7), 1746-1758.

Publication 2014

Von Willebrand Factor integrin α6 CD133 proliferating cell nuclear antigen phospho histone H3 SP-5 confocal microscope

Anatomical regions Antibodies Blood vessels Brains Confocal microscope Frozen Histone h3 Human Immunofluorescence Integrin Knockout mice Mice Normal tissue Novus Nuclei Paraformaldehyde Proliferating cell nuclear antigen Sucrose Von willebrand factor Xenografts

Corresponding Organization : Cleveland Clinic Lerner College of Medicine

Other organizations : University of Kentucky, Medical College of Wisconsin, Neurological Surgery

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Variable analysis

independent variables

Genotype (wild-type vs. CD36 knockout)

dependent variables

Cell proliferation (measured by PCNA and phospho-histone H3 staining)
Blood vessel visualization (measured by Von Willebrand Factor staining)
Expression of cell surface markers (CD36, integrin α6, CD133, Iba1)

control variables

Tissue preparation (snap freezing for human samples, paraformaldehyde fixation, sucrose incubation, and OCT embedding for xenografts and mouse brains)
Sectioning thickness (10 μm)
Immunofluorescence staining and imaging (Dapi nuclear counterstaining, Leica SP-5 confocal microscope)

controls

Positive control: Age-matched wild-type mouse brains
Negative control: CD36 knockout mouse brains

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