Rabbit anti enos

Three days after injection with LSS- or OSS-sEVs, mouse carotid arteries, LCA, and RCA were collected and fixed in 4% paraformaldehyde (Biosesang Inc., Seongnam, Korea) for 20 min at room temperature. After permeabilization in 0.05% Triton X-100 (in PBS) for 20 min and blocking in 10% donkey animal serum for 1 h at room temperature, carotid arteries were incubated with rabbit anti-eNOS (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-VCAM-1 (1:200, Abcam, Cambridge, UK), or rat anti-ICAM-1 (1:500, Southern Biotech) antibody overnight at 4 °C. After washing in PBS three times, carotid arteries were incubated with Alexa Fluor 568-conjugated donkey anti-rabbit (1:500, Invitrogen, Carlsbad, CA, USA) IgG or Rhodamine Red-X (RRX) goat anti-rat IgG (1:500, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) secondary antibody for 2 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 200 ng/mL; Santa Cruz Biotechnology, Dallas, TX, USA) for 5 min. We detected the fluorescence signals using a Zeiss LSM 800 confocal microscope.

G-CSF was provided by UBI pharma Inc. (Taipei, Taiwan). Fluorescein isothiocyanate (FITC) anti-mouse Sca-1 and phycoerythrin (PE) anti-mouse Flk-1 antibodies were purchased from eBioscience (San Diego, CA, USA). Isotype-identical antibodies were obtained from Becton Dickinson (Franklin Lakes, NJ, USA). The antibodies listed below were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): rabbit anti-eNOS, anti-p-eNOS, mouse anti-VEGF, anti-α-SMA, and anti-β-actin. Rabbit antibodies for STAT3 and p-STAT3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Mouse antibodies for IL-10 were purchased from Serotec (Oxford, UK). Rat anti-CD31 was obtained from BD Pharmingen (San Diego, CA, USA). Phenylmethylsulfonyl fluoride (PMSF) protease inhibitor, Histopaque-1077 (density: 1.077 g/mL), and 2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Endothelial growth medium (EGM-2 MV) and growth factors for EPC culture were obtained from Lonza (Morristown, NJ, USA).