Anti cd34 primary antibody

Myocardial tissue sections were stained with anti-CD34 primary antibody (1 : 200, Abcam, Cambridge, UK). Vascular endothelial cells were immunohistochemically labeled with CD34 to calculate microvessel density (MVD) [21 (link)], which was quantified by counting the number of brown endothelial cells or endothelial cell clusters. Five microscopic fields were randomly selected for each tissue section under 400× magnification, and their average values were taken as the MVD of each sample.

Mice were anesthetized and skull burr-hole drilled. Animals were each focally injected with 5 × 10³ MDA-MB-231 tumor cells expressing empty vector (pCDNA3) or MYC-RASSF1C in 0.5 μl PBS in the left striatum using a 75-mm-tipped glass microcapillary (Clark Electromedical Instruments). At day 21, all animals were transcardially perfusion fixed under terminal anesthesia (n = 4 per group) and brains were post-fixed, cryoprotected, embedded, and frozen in isopentane at −40°C. To assess areas of tumor colonization, photomicrographs of each brain section were obtained using ScanScope CS slide scanner (Aperio) and analyzed using ImageScope (Aperio). For immunofluorescence, sections were streptavidin and biotin blocked, incubated with anti-CD34 primary antibody (Abcam; brain vessels) or anti-vimentin antibody (VectorLabs; tumor cells), washed, and incubated with a streptavidin-Cy3 fluorophore or AMCA-conjugated secondary antibody (Invitrogen; 1:100) for 30 min. Expanded animal experimental procedures are outlined in Supplemental Experimental Procedures.
All animal experiments were approved by the University of Oxford local animal ethical committee and were performed according to terms of a license granted by the UK Home Office, adhering to the Animals (Scientific Procedures) Act 1986.

Qualitative and quantitative analysis of VM in nude mouse tumor xenograft tissue sections was detected by CD34-PAS assay [8 (link)]. The embedded nude mouse tissues were sectioned and then dewaxed, hydrated and antigenically repaired. Tissue specimens were blocked with goat serum and incubated overnight at 4°C with anti-CD34 primary antibody (1:200, #81289, Abcam, USA). Then, incubated with anti-mouse HRP-labeled polymer secondary antibody at 37°C for 30 min. Finally, DAB (Proteintech, China) was added for staining, followed by staining using the Glycogen PAS staining solution set (Dalian Meilun Biotechnology Co., Ltd., China). VM quantification was performed on at least three random regions using a fluorescence microscope DMi8 (Leica, Germany).

Human bone marrow derived CD34+ cells were cultured in fibronectin‐coated (1 μg/cm²) tissue culture flasks at 37°C in EBM‐2 with endothelial growth medium‐2 SingleQuots™ Supplements. Medium was changed after 3 days. Cell colonies appeared after 7 days of culture. To confirm identification of ECFCs the cells were characterized by immunostaining of CXCR4 (CXCR4, receptor specific for SDF‐1α) and CD34 (marker of vascular endothelial progenitor cells) (Images not shown). ECFCs were plated at 5000 cells/cm² and incubated for 24 h. Then cells were fixed with 4% paraformaldehyde (PFA) for 15 min and blocked with 1% BSA overnight at 4°C. Cells were incubated with rabbit anti‐CXCR4 primary antibody from Thermo Scientific (1:200, Waltham, MA) or with anti‐CD34 primary antibody Abcam (1:100, Cambridge, MA) for 1 h at 37°C, and then incubated with AlexaFluor 488 goat antirabbit IgG secondary antibody from Abcam (1:1000, Cambridge, MA) for 2 h at 37°C. Cell nuclei were stained with DAPI.

Immunohistochemical analysis was conducted on the specimens with bone formation detected by histology. After incubating with the anti-CD34 primary antibody (Abcam, UK, 1:500), the secondary antibody was added. StreptABComplex/HRP (Dako Corp., USA) was then employed to amplify the signals of cells labeled with diaminobenzidine (Dako, USA).