Immunofluorescence Analysis of Cardiac Tissue

Immunofluorescence was performed on 5-μm thick formalin-fixed and OCT-embedded heart sections. Briefly, formalin-fixed paraffin embedded sections were deparaffinized, followed by antigen retrieval and blocking with blocking buffer (1% BSA in 1× PBS) for 1 h [20 (link)]. Similarly, OCT-embedded sections were fixed with 4% paraformaldehyde for 20 min and rehydrated in 1× PBS for 30 min. Sections were then incubated with primary antibody against rat anti-mouse neutrophil (Serotec), rat anti-mouse F4/80 (Serotec), mouse anti-nitrotyrosine (anti-NT) (Santa Cruz Biotechnology), mouse anti-4-hydroxynonenal (anti-4-HNE) (Abcam), overnight in a humidified chamber at 4°C. Sections were incubated with different fluorophore-conjugated secondary antibodies (Invitrogen, U.S.A.) as described recently [20 (link)].