Nitrotyrosine

As previously described^{18 (link)}, kidney samples were run on polyacrylamide minigels. After transfer by electroelution to polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK), blots were blocked with 5% nonfat dry milk in Tris-buffered saline. Blots were then incubated overnight with antibodies against caspase 3 (1:500), heme oxygenase-1 (HO-1, 1:1,000), peroxiredoxin 6 (Prdx6, 1:1,000), and nitrotyrosine (1:1,000), all of which were obtained from Abcam (Cambridge, UK); and against glutathione peroxidase 4 (Gpx4, 1:1,000), obtained from Cell Signaling Technology (Danvers, MA, USA). We visualized the labeling with a horseradish peroxidase-conjugated secondary antibody, using enhanced chemiluminescence detection (Amersham Pharmacia Biotech, Piscataway, NJ, USA). We scanned the films with an imaging system (Alliance 4.2; UVItec, Cambridge, UK), after which we used densitometry to perform a quantitative analysis of the antibodies employed, normalizing the bands to β-actin expression.

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.

Protein samples (10 to 40 μg) were subjected to SDS-PAGE and transferred to Immobilon P membranes (Millipore Corp, Bedford, MA). After blocking, membranes were probed with primary antibodies against αSMA (1:1000; Dako), nitrotyrosine (NT, 1:1000; Abcam), or GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies at 1: 2000 dilutions. Immunoreactive bands were visualised using the ECL detecting reagent (GE Healthcare UK Ltd, Buckinghamshire, UK) and documented with a Fujifilm Image Reader LAS-3000 (Fujifilm, Tokyo, Japan) coupled to image analysis software (Multi Gauge, Fujifilm).