LV myocardial tissue samples were fixed in buffered paraformaldehyde solution (4%) and embedded in paraffin or stored in -80°C until they could be cut into frozen sections. Then, blocks were cut into 5-μm-thick paraffin or frozen sections. The immunoreactivity to myeloperoxidase (MPO) (1:100; Abcam, Cambridge, UK) and nitrotyrosine (1:100; Abcam, Cambridge, UK) was investigated. Infiltrating neutrophils (MPO-labeled) were counted, and nitrotyrosine expression was semi-quantitatively assessed based on staining intensity and the distribution of the labelled target protein. Furthermore, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining to detect DNA-strand breaks as described previously [13 (link)–15 (link)]. The number of TUNEL-positive cells was expressed as the ratio of DAPI-TUNEL double-labeled nuclei to the total number of nuclei stained with 4’, 6-diamidino-2-phenylindole (DAPI). Each specimen recieved an average score of four adjacent fields in a blinded fashion.
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