Mouse anti 4 hne

Immunofluorescence was performed on formalin-fixed paraffin-embedded heart sections (5 µm). Briefly, the sections were deparaffinized, followed by antigen retrieval and blocked with blocking buffer (1% BSA in 1X PBS) for 1 h. Similarly, OCT-embedded sections were fixed with 4% paraformaldehyde for 20 min and rehydrated in PBS for 30 min. Then, sections were incubated overnight in a humidified chamber at 4°C with primary antibody against rat anti-mouse neutrophil (Serotec), rat anti-mouse F4/80 (Serotec), mouse anti-nitrotyrosine (Santa Cruz), mouse anti-4-HNE (Abcam). Finally, sections were incubated with different fluorophore-conjugated secondary antibodies (Invitrogen USA), as described previously (26 (link), 32 (link)).

Proteins were isolated from snap frozen liver tissue by homogenising in ice-cold Tris-HCl (50 mM, pH 7.6) with protease inhibitor cocktail (Sigma-Aldrich Company Ltd., Gillingham, UK), PMSF, and EDTA. The protein concentration was determined using Bradford reagent. Cultured cells were lysed in ice-cold RIPA buffer. The protein concentration was measured using a BCA assay kit (Life Technologies Ltd., Paisley, UK). Equal amounts of protein were electrophoresed through 4–12% NuPAGE Bis-Tris Gels and transferred to PVDF membranes (Life Technologies Ltd., Paisley, UK). Blots were probed with the following primary antibodies: rabbit anti-DDAH1 (Abcam, Cambridge, UK); mouse anti-eNOS (BD, Oxford, UK), mouse anti-NF-κB, (Cell Signalling Technology, Danvers, MA, USA), mouse anti-4HNE (Abcam, Cambridge, UK), rabbit anti-Vegf-A (Santa Cruz Biotechnology, Inc, Dallas, TX, USA), mouse anti-Gapdh (loading control; Abcam, Cambridge, UK), and mouse anti-α-tubulin (loading control; Millipore, Watford, UK). Immune complexes were detected using HRP-conjugated secondary antibodies (Cell Signalling Technology, Danvers, MA, USA) and enhanced chemiluminescence (ECL) reagents (GE Healthcare Lifesciences, Little Chalfont, UK). Densitometric quantification was performed using Image J (US NIH, Bethesda, MD, USA; http://imagej.nih.gov/ij/).