Paraffin-embedded tissue sections received a classical resorcin-fuchsin (RF) stain (Sigma).
Immunohistochemistry was performed on paraffin-embedded tissue sections of the thoracic aorta against 3-nitrotyrosine (NT). After deparaffinization, antigens were retrieved by heating the slides in citrate buffer (pH = 3). We blocked endogenous peroxidase activity with 3% H2O2 in dH2O2. Nonspecific labeling was evitable using 2.5% normal horse serum (Vector Biolabs, Burlingame, CA, USA). After overnight application of primary antibodies (polyclonal rabbit anti-NT 1 : 500, Merck Millipore, Burlington, MA, USA) at 4°C, horseradish-peroxidase- (HRP-) linked anti-mouse monoclonal horse antibodies (Vector Biolabs) provided secondary labeling which was visualized by brown-colored diamino-benzidine (DAB, Vector Biolabs). For counterstaining, blue-colored hematoxylin (Vector Biolabs) was utilized. Zeiss Axio Imager system (Zeiss, Oberkochen, Germany) was used for microscopic imaging of tissue sections. Uncalibrated optical density of brown coloring was estimated by ImageJ software (NIH, Bethesda, MA, USA).
Immunohistochemistry was performed on paraffin-embedded tissue sections of the thoracic aorta against 3-nitrotyrosine (NT). After deparaffinization, antigens were retrieved by heating the slides in citrate buffer (pH = 3). We blocked endogenous peroxidase activity with 3% H2O2 in dH2O2. Nonspecific labeling was evitable using 2.5% normal horse serum (Vector Biolabs, Burlingame, CA, USA). After overnight application of primary antibodies (polyclonal rabbit anti-NT 1 : 500, Merck Millipore, Burlington, MA, USA) at 4°C, horseradish-peroxidase- (HRP-) linked anti-mouse monoclonal horse antibodies (Vector Biolabs) provided secondary labeling which was visualized by brown-colored diamino-benzidine (DAB, Vector Biolabs). For counterstaining, blue-colored hematoxylin (Vector Biolabs) was utilized. Zeiss Axio Imager system (Zeiss, Oberkochen, Germany) was used for microscopic imaging of tissue sections. Uncalibrated optical density of brown coloring was estimated by ImageJ software (NIH, Bethesda, MA, USA).