goat anti mouse vegfr2 polyclonal antibody  (R&D Systems)

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Confocal micrographs of projected optical sections of native EDL muscle (top row, 20-μm-thick projection) and EDL muscle 5 days after ischemic injury (bottom row, 16 μm thick), immunostained for CD31 (green), VEGFR2 (grayscale), p-VEGFR2 (red), and DAPI (blue). Arrows depict the borders of a mother vessel with several microdivisions/early splitting zones (asterisks). Strong p-VEGFR2 immunoreactivity is present in the primordial vessels but not capillaries. ( B ) Epifluorescence micrographs of transverse sections of EDL muscle 5 days after ischemic injury and 16 hours after receiving vehicle (left) or apatinib (right), immunostained for CD31, p-VEGFR2, and DAPI. p-VEGFR2 signals are diminished in CD31-positive microvessels and nonvascular cells in mice receiving apatinib. ( C ) RBC transit maps of microvascular networks in regenerating (5-day) EDL muscle 16 hours after receiving apatinib, cabozantinib or ZM323881 (small-molecule inhibitors of VEGFR2) or DC101 (VEGFR2-blocking antibody), and respective controls. Pillars have been circled yellow (arrows) and microdivisions/splits are outlined in dashed blue lines. Graphs depicting the density of microvascular networks are shown on the right [means ± SE; n = 26/13, 17/12, 17/19, and 21/18 distinct muscle territories (0.63 mm 2 ) in the EDL subjected to control or designated blocking agent]. The effects of cabozantinib and ZM323881 were studied using the same vehicle controls [dimethyl sulfoxide/polyethylene glycol (PEG)/saline], and P values are Bonferroni corrected. ( D and E ) Graphs depicting the density of pillars (D) and splits (E) in EDL muscle 5 days after ischemic injury. Data are expressed relative to the respective controls, depicted by the dashed line (means ± SE, n = 13, 12, 19, and 18).

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Blocking Assay, Control, Saline

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Confocal micrographs of planar-projected optical sections of a primordial vessel in EDL muscle 5 days after ischemic injury immunostained for CD31 (green) and p-(activated) VEGFR2 (red), with TO-PRO-3–stained nuclei. A CD31-expressing pillar is present (arrow). There is abundant p-VEGFR2 signal in endothelial cells of the wall but not the pillar. ( B ) Graph depicting p-VEGFR2 signals in endothelial pillars versus wall-lining endothelium of the same vessel. N = 8 pillars from different primordial vessels. Wall and pillar endothelial cell data from a given primordial vessel are connected. A.U., arbitrary units. ( C ) Confocal planar projection of a primordial vessel showing a CD31-expressing pillar endothelial cell (arrow) with modest p-VEGFR2 signal lateralized to one face, orthogonal to the vessel long axis (small arrows). ( D ) Confocal orthogonal (XZ, YZ) reconstructions of a day 5 primordial vessel showing an endothelial cell pillar structure inside the lumen. p-VEGFR2 signal is abundant in the lining endothelium, but on the pillar p-VEGFR2 appears as a discrete lateralized signal. ( E ) Distribution of p-VEGFR2–positive pillars with diffuse versus lateralized p-VEGFR2 signal. ( F ) Confocal planar projections through a pillar within a primordial vessel in day 5–regenerated muscle showing VEGFR2 activity that is focal, lateralized, and enriched at the midzone of the pillar (arrows). The two planar projections are 3 μm apart. A schematic of the micrograph is depicted (right). ( G ) Distribution of p-VEGFR2 locations in lumen-crossing pillars.

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Staining, Expressing, Activity Assay

Journal: Science Advances

Article Title: Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle

doi: 10.1126/sciadv.abg9509

Figure Lengend Snippet: ( A ) Graph depicting the aspect ratio of control and VEGFR2 knockdown endothelial cells lining a PDMS microfluidic device subjected to ultralow shear stress (0.3 dyne/cm 2 ). A total of 394 cells with control siRNA and 379 with VEGFR2 siRNA from four different devices were quantified. Median values and all data points are shown. ( B ) Pillar content, defined as the proportion of endothelial cells at microchannel shoulders that formed a pillar, for differentially labeled control and VEGFR2 knockdown endothelial cells. Data are from four independent experiments and depicted as mean and SE. ( C to E ) Confocal microscopy volume projections of differentially labeled control and VEGFR2 knockdown endothelial cells lining a microfluidic device and subjected to ultralow shear stress. In (C) and (D), control-siRNA cells express GFP, and VEGFR2-siRNA cells express RFP. In (E), the labeling is reversed. Nuclei were stained with DAPI. In (C) and (E), pillars are composed of VEGFR2-siRNA cell projections that span across the lumen and connect with a control-siRNA cell on the opposite wall. In (D), a VEGFR2-siRNA cell body crosses the lumen and is connected on either side with control siRNA cells lining the device walls. ( F ) Pillar content of microfluidic channels lined by HUVECs subjected to ultralow shear stress (0.3 dyne/cm 2 ) and infused with L-NAME (10 μM) or water vehicle. ( G ) Pillar content of endothelial cell–lined microchannels under ultralow shear conditions infused with DETA NONOate (1 μM), ZM323881 (10 nM), or the combined interventions. Data are means and SE.

Article Snippet: Triple immunostaining was performed using rat monoclonal anti-CD31 antibody (1:20; Clone SZ31, Dianova), rabbit polyclonal anti–p-VEGFR2 Y1054/1059 antibody, and goat polyclonal anti-VEGFR2 antibody (1:100; AF644, R&D Systems).

Techniques: Control, Knockdown, Shear, Labeling, Confocal Microscopy, Staining