Mouse anti α sarcomeric actin α sa

Both canine MSCs and CDCs were cultured in the following differentiation media for 12 days: (i) cardiomyocyte differentiation media: IMDM with 1% N2 (Cat no. 12440; Gibco, Carlsbad, CA, USA), and 100 ng/ml Heregulin-β1 (Cat no. 100-03; Peprotech, Rocky Hill, NJ, USA); (ii) smooth muscle differentiation media: IMDM with 10 ng/ml platelet-derived growth factor-beta (Cat no. 100-14B; Peprotech) and (iii) endothelial differentiation media: IMDM with 50 ng/mL VEGF (Cat no. 100-20A; Peprotech) 18 . After the differentiation process, the cells were fixed with 4% PFA, blocked/permeabilized with Protein Block Solution (DAKO) containing 1% saponin (Sigma-Aldrich), and then stained with mouse anti- α-sarcomeric actin (α-SA) (Sigma-Aldrich), mouse anti-smooth muscle actin (Sigma-Aldrich) and rabbit anti-von Willebrand factor (Abcam) antibodies. FITC or Texas-Red secondary antibodies were obtained from Abcam as well. Cell nuclei were counter-stained with 4’,6-diamidino-2-phenylindole (DAPI).

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.).