Immunohistochemical Analysis of Nitrotyrosine

The nitrotyrosine content was also measured by immunohistochemistry staining. The detailed protocol was described in our previous publications [21 (link), 22 (link)]. The LV tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Then, they were cut into 3 mm thickness and stained with primary antibody (anti-nitrotyrosine antibody, Cell Signaling Technology, MA, USA, 1 : 200 dilution). Then, the sections were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (Zhongshan Biotechnology, Beijing, China) and detected with 3,3′-diaminobenzidine (DAB) staining (Zhongshan Biotechnology, Beijing, China). Five fields of each section were randomly chosen and photographed at ×200 magnification (Olympus BX-63, Tokyo, Japan). The graphs were analyzed and calculated using Image-Pro Plus software (Media Cybernetics, MA, USA).