Mouse/Rat Tie-2 Antibody

Tie-2 in Mouse Intestine Tissue. Tie-2 was detected in immersion fixed paraffin-embedded sections of mouse intestine tissue using Goat Anti-Mouse/Rat Tie-2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF762) at 1 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC004). Before incubation with the primary antibody, tissue was subjected to heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (Catalog # CTS013). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cell surface on endothelial cells. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.

Detection of Mouse Tie-2 by Western Blot Pericytes express functional Tie2.(a) Microarray-based expression screening of brain pericytes (BP), placenta pericytes (PP), pancreas pericytes (PA), lung pericytes (LP), muscle pericytes (MP) and human umbilical vein endothelial cells (HUVEC [HU]). (b) Semi-quantitative PCR of TEK (Tie2), endothelial marker genes (PECAM1, CDH5) and pericyte marker genes (CSPG4, CD248) in HUVEC and BP; house keeping gene: GAPDH. (c) Representative images showing expression of Tie2 in HUVEC and BP. (d) Histogram of membrane-bound Tie2 expression in HUVEC and BP compared to isotype control measured by flow cytometry. (e,g) Western blot (WB) analysis of tyrosine phosphorylation (pTyr) and total Tie2 after Tie2 immunoprecipitation (IP) in BP (e) and HUVEC (g) upon stimulation with recombinant human (rh) Ang1 compared to unstimulated (us) cells. (f,h) Quantification of the ratio of phosphorylated Tie2 (pTie2) relative to total Tie2 protein in BP (f) and HUVEC (h) normalized to us control (n=3). (i,k) Western blot analysis of AKT phosphorylation (Ser473) in control (shCtr) and Tie2-silenced (shTie2 I/shTie2 II) BP (i) and HUVEC (k) upon stimulation with rhAng1 or rhAng2. (j,l) Quantification of the ratio of pAKT to total AKT protein expression in BP (j) and HUVEC (l) normalized to unstimulated control (n=3). Representative western blot images are cropped versions and original images can be found in Supplementary Fig. 15. Scale bars: 20 μm (c). Data are shown as mean±s.d. Statistics were performed using Mann–Whitney U test (f,h) and one-way ANOVA (j,l). *P<0.05, **P<0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28719590), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Tie-2 by Immunocytochemistry/ Immunofluorescence Endothelial changes after pericyte depletion. a–f Maximum intensity projection of confocal images from control and DTRiPC P6 retinas stained for IB4 (red) in combination with VEGF-A a, VEGFR2 b, VEGFR3 c, Tie2 d, Esm1 e, and Dll4 f (all in white), as indicated. Note local increase of VEGFR2, VEGFR3, and Esm1 (arrowheads in b, c, e) but not Tie2 or VEGF-A at the edge of the vessel plexus. Dll4 expression in DTRiPC sprouts is increased in some regions (arrowheads) but absent in others (arrows). Scale bar, 100 µm. g–j Quantitation of VEGF-A immunosignals area and intensity g, signal intensity for VEGFR2 h and VEGFR3 i and proportion of Esm1+ area with respect to vascular area j in the P6 control and DTRiPC angiogenic front. Error bars, s.e.m. p-values, Student’s t-test. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29146905), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Tie-2 by Western Blot Pericyte-derived Angpt1 controls alveologenesis. a RT-qPCR analysis of Angpt1 and Tie2/Tek expression in freshly sorted lung GFP+, CD31+ or EpCAM+ cells from P7 Pdgfrb(BAC)-CreERT2 R26-mT/mG mice. Data represents mean ± s.e.m. (n = 4 mice). b High magnification images of P10 Angpt1GFP lungs stained for GFP (green), PDGFR beta (red), and PDGFR alpha (blue). Arrows indicate GFP and PDGFR beta double positive pericytes. Scale bar, 15 µm. c RT-qPCR analysis of Angpt1 expression in freshly sorted PDGFR beta + cells from P7 Yap1,Wwtr1iPCKO and control lungs. Data represents mean ± s.e.m. (n = 4 mice, two-tailed unpaired t-test). dAngpt1 expression in cultured Verteporfin (VP)-treated (48 h) and control pericytes. Data represents mean ± s.e.m. (n = 4, Welch’s t-test). e Expression of the indicated transcripts in freshly sorted CD31+ cells from P7 Yap1,Wwtr1iPCKO and control lungs. Data represents mean ± s.e.m. (n = 4 mice, NS not significant, two-tailed unpaired t-test). f–h Western blot analysis of Angpt1 protein (f; n = 2 controls and 4 mutant mice) and of total and phospho-Tie2 (pTie2) in P12 Yap1,Wwtr1iPCKO and control total lung lysates (g, n = 3 controls and 5 mutants). Molecular weight marker (kDa) is indicated. Relative quantification of signals is shown in h. Two-tailed unpaired t-test. i Scheme showing the time points of tamoxifen administration and analysis for Angpt1iPCKO mice. j, k 3D reconstruction confocal images of P12 Angpt1iPCKO and littermate control lungs stained for AQP5 (green), PDGFR beta (red), and PECAM1 (blue). Panels in k show higher magnification of PECAM1 staining. Scale bar, 50 µm (j) and 30 µm (k). l Quantitation of airspace volume in P12 Angpt1iPCKO and littermate control lung sections with 3D reconstruction surface images. Data represents mean ± s.e.m. (n = 4 mice; p < 0.0001, two-tailed unpaired t-test). m 3D reconstruction confocal images of P12 Angpt1iPCKO and littermate control lungs stained for alpha SMA (red) and tropoelastin (blue). Scale bar, 50 µm. n Quantitation of staining intensity for alpha SMA or tropoelastin shown in m. Intensity was normalized to the size of the parenchymal region. Data represents mean ± s.e.m. (n = 4 mice; NS not significant, two-tailed unpaired t-test). o Schematic summary of findings. Pulmonary pericytes regulate ECs and alveolar epithelial cells via angiocrine factors such as angiopoietin-1 and HGF. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/29934496), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Tie-2 by Western Blot Pericytes express functional Tie2.(a) Microarray-based expression screening of brain pericytes (BP), placenta pericytes (PP), pancreas pericytes (PA), lung pericytes (LP), muscle pericytes (MP) and human umbilical vein endothelial cells (HUVEC [HU]). (b) Semi-quantitative PCR of TEK (Tie2), endothelial marker genes (PECAM1, CDH5) and pericyte marker genes (CSPG4, CD248) in HUVEC and BP; house keeping gene: GAPDH. (c) Representative images showing expression of Tie2 in HUVEC and BP. (d) Histogram of membrane-bound Tie2 expression in HUVEC and BP compared to isotype control measured by flow cytometry. (e,g) Western blot (WB) analysis of tyrosine phosphorylation (pTyr) and total Tie2 after Tie2 immunoprecipitation (IP) in BP (e) and HUVEC (g) upon stimulation with recombinant human (rh) Ang1 compared to unstimulated (us) cells. (f,h) Quantification of the ratio of phosphorylated Tie2 (pTie2) relative to total Tie2 protein in BP (f) and HUVEC (h) normalized to us control (n=3). (i,k) Western blot analysis of AKT phosphorylation (Ser473) in control (shCtr) and Tie2-silenced (shTie2 I/shTie2 II) BP (i) and HUVEC (k) upon stimulation with rhAng1 or rhAng2. (j,l) Quantification of the ratio of pAKT to total AKT protein expression in BP (j) and HUVEC (l) normalized to unstimulated control (n=3). Representative western blot images are cropped versions and original images can be found in Supplementary Fig. 15. Scale bars: 20 μm (c). Data are shown as mean±s.d. Statistics were performed using Mann–Whitney U test (f,h) and one-way ANOVA (j,l). *P<0.05, **P<0.01. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28719590), licensed under a CC-BY license. Not internally tested by R&D Systems.