Systemic oxidative stress was assessed by IVC levels of 8-epi-isoprostane (EIA kit) [23 (link)]. Myocardial oxidative stress was assessed by in-situ production of superoxide anion with dihydroethidium (DHE) and by expression of the NADPH-oxidase subunits p47 and p67 (1 : 200; both Santa Cruz), and by nitrotyrosine (1 : 200; Cayman Chemical Company, Ann Arbor, Michigan, USA) [23 (link),24 (link)].
Inflammation was evaluated in myocardial sections by standard immunostaining for MCP-1 (1 : 7500; MyBioSource Inc., San Diego, California, USA) and double staining for pro-inflammatory CD68+/iNOS+ (M1) (Abnova Inc., Walnut, California, USA; catalog#: ab15323, 1 : 100) and reparative CD68+/Arinase-1 (M2) (sc-20150 cat#: HPA004114, 1 : 100) macrophages. In addition, myocardial expression of tumor necrosis factor (TNF)-α (Santa Cruz, 1 : 200), interleukin (IL)-6 (1 : 500; Abcam), and IL-10 (1 : 200; Santa Cruz) was quantified by western blot.
Inflammation was evaluated in myocardial sections by standard immunostaining for MCP-1 (1 : 7500; MyBioSource Inc., San Diego, California, USA) and double staining for pro-inflammatory CD68+/iNOS+ (M1) (Abnova Inc., Walnut, California, USA; catalog#: ab15323, 1 : 100) and reparative CD68+/Arinase-1 (M2) (sc-20150 cat#: HPA004114, 1 : 100) macrophages. In addition, myocardial expression of tumor necrosis factor (TNF)-α (Santa Cruz, 1 : 200), interleukin (IL)-6 (1 : 500; Abcam), and IL-10 (1 : 200; Santa Cruz) was quantified by western blot.
