Immunohistochemical Analysis of Myocardial MMP-9

Matrix metalloproteinase (MMP)-9 is a proximal biomarker for cardiac remodelling and angiogenesis. The expression of myocardial MMP-9 was examined with immunohistochemical analyses as previous reported 21 (link). Hearts were fixed with formalin, embedded with paraffin and cut to obtain 5 μm sections. After incubation of sections for 10 min. with 0.3% H₂O₂, a serum-free protein block (DAKO, Carpinteria, CA, USA) was added for 10 min. Before adding MMP-9 primary antibody, the slides were treated with monohydrated citrate buffer (pH 6.0, 0.01 M) in a water bath for 10 min. at 100°C for the antigen retrieval. Sections were then incubated with the monoclonal antibodies against MMP-9 (1:100; Calbiochem®, San Diego, CA, USA) for 1 hour at room temperature. Anti-MMP-9 recognizes both latent and active form. Non-immune mouse serum was substituted for negative controls. After incubation for 10 min. with a biotinylated secondary antibody, AEC chromogen (DAKO) was used to develop the horseradish peroxidase-streptavidin complex.