Anti pecam

Total protein concentrations of supernatant fractions were determined using the bicinchoninic acid (BCA) protein assay (Bio-Rad, Inc.). Equal amounts of protein aliquots were boiled in equal volumes of 2× SDS Laemmli sample buffer and resolved on 8% or 10% (w/v) with primary antibodies; anti-NOTCH1 (0.2μg/ml; Abcam^®, Inc.), anti-Dll4 (0.1μg/ml; Abcam^®, Inc.), anti-LYVE-1 (1μg/ml; Abcam^®, Inc.) and anti-PECAM (2μg/ml; Abcam^®, Inc.). Immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using the enhanced chemiluminescence technique.

Cell cultures were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 20 min, washed, and permeabilized with PBS containing 0.1% Triton X-100 (Fisher Scientific) and 4% normal goat serum (Jackson ImmunoResearch Labs, West Grove, PA, USA) for 20 min, followed by the incubation of primary antibodies overnight at 4 °C. After three 10 min PBS washes; the cells were incubated with secondary antibodies for 1 h at room temperature followed by extensive washes. The antibodies included: anti-Nestin (mouse clone 10C2, 1:100, eBioscience), anti-Ki67 (mouse clone B56, 1:100, BD Biosciences), anti-glial fibrillary acidic protein (GFAP, mouse clone GA5, 1:500, eBioscience), anti-Vimentin (mouse clone RV202, 1:200, BD Bioscience), anti-alpha smooth muscle actin (SmA, rabbit clone E184, 1:100, Abcam, Cambridge, MA USA), and anti-PECAM (rabbit, 1:100, Abcam). Goat anti-mouse or rabbit Alexa 488 and 568 (1:250; Invitrogen) secondary antibodies were used. Fluorescence images were acquired on a Leica DM IL fluorescence microscope using an excitation/emission (Ex/Em) of 470/525 nm for Alexa 488 and Ex/Em of 560/645 nm for Alexa 568. Confocal images were acquired on a Zeiss 780 laser scanning confocal imaging system.

Tissues were fixed in 4% paraformaldehyde and decalcified in 10% EDTA. Paraffin sections were stained with Masson’s trichrome stain for morphological analysis. For IHC, antigens of de-paraffinized sections were retrieved by 0.05% trypsin or hyaluronidase (10 mg/ml) and treated with 3% H₂O₂. After blocking with 5% normal goat serum, tissues were incubated with primary antibodies in 4°C, overnight. The following rabbit polyclonal antibodies against mouse were used, anti-VEGF (Abcam, Cambridge, UK), anti-PECAM (Abcam), and anti-Osteocalcin (Millipore, Billerica, MA, USA). Sections were then incubated with anti-rabbit secondary antibody (Vectastain ABC system, Vector Laboratories, Servion, Switzerland) and developed with 0.1% 3, 39-diaminobenzidine. Images were captured using standard light microscopy (Zeiss, Oberkochen, Germany) and quantified using Image-Pro Plus software (Rockville, MD, USA). Data from three independent mice staining were used for statistical analysis.