Goat anti human lyve 1

LECs were treated with human TGF-β1, -β2 and β3 for the indicated time points. Lysates were prepared and subjected to Western blot analysis using 5 μg/ml polyclonal rabbit-anti-human Prox-1 (Reliatech, Cat. No. 102-PA32AG), 1 μg/ml polyclonal goat-anti-human Lyve-1 (R&D Systems, Cat. No. AF2089), 0.2 μg/ml polyclonal goat-anti-human VEGF-R3 (R&D Systems, Cat. No. AF349) and 0.1 μg/ml polyclonal goat-anti-human vimentin (R&D Systems, Cat. No. AF2105) antibodies. For evaluation of protein loading, the blot was probed with 0.01 μg/ml monoclonal mouse-anti-human vinculin-specific antibodies (Sigma Aldrich, Taufkirchen, Germany, Cat. No. V9264). For densitometric evaluation, bands for the Prox-1, Lyve-1, vimentin and VEGFR-3 proteins were normalized to the corresponding loading control using the software ImageJ. Values are presented relative to untreated control samples.

Four-μm-thick histologic sections of the myocardium were deparaffinized in xylene, rehydrated, and subjected to 10 min heat-induced antigen retrieval in citric buffer (pH 6.0). Samples were blocked in 10% normal donkey serum (Jackson ImmunoResearch Inc., West Grove, PA) for 30 min at room temperature, incubated with 10 μg/ml of LpMab-12 overnight at 4°C, and then with Alexa Fluor 568-conjugated donkey anti-mouse IgG (Thermo Fisher Scientific Inc.) for 1 h at 37°C. Subsequently, the sections were incubated with goat anti-human LYVE-1 (10 μg/ml; R&D Systems, Inc., Minneapolis, MN) and mouse anti-α-sarcomeric actin (α-SA) (1:200 diluted; Sigma-Aldrich Corp.) for 2 h at 37°C, followed by fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG and Alexa Fluor 647-conjugated donkey anti-mouse IgM (15 μg/ml each; Jackson ImmunoResearch Inc.) and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (1 μg/ml; Sigma-Aldrich Corp.) for 1 h at 37°C. The sections were then treated with 1% solution of Sudan Black B (Sigma-Aldrich Corp.) for 30 min at room temperature, and mounted in Vectashield medium (Vector Laboratories, Inc., Road Burlingame, CA). Images were acquired with Olympus FluoView FV100 laser scanning confocal microscope equipped with CCD camera (Bio-Rad Laboratories Inc.).