2.4 Immunohistochemistry
Presence and distribution of NOS, SOD1 and catalase were determined in arteries from a subset of 28 randomly selected patients. Four μm thick cross-sections of the formalin-fixed paraffin embedded arterial segments were obtained and mounted on SuperFrost Plus Adhesion slides (Thermo Fisher Scientific, Waltham, MA, USA). Samples were fixed on the glass slides at 60°C for 30 min and then deparaffinized and rehydrated through a series of Tissue-Clear (Sakura, Alphen aan den Rijn, The Netherlands), and graded ethanol (99-70%, Merck). Antigens were retrieved by heating in sodium citrate buffer (10 mM, pH6, Sigma-Aldrich) at 97⁰C in an oven for 22 min. and then cooled on ice for 25 min. Blocking of endogenous peroxidase was performed in 0.3% H2O2and 50 mM NH4Cl in PBS for 10 min at RT. Blocking was performed using PBS/0.025% Triton for 30 min. Primary antibodies were diluted and applied in the blocking buffer as follows; CAT (1:1000, ab52477, Abcam, Cambridge, UK), eNOS (1:100, PA1-037, InvitrogenTM via Fischer Scientific, Roskilde, Denmark), iNOS (1:300, PA1-036, Fischer Sci), nNOS (1:400, ab1376, Abcam) and SOD1 (1:400, MA1-105, InvitrogenTM via Fischer Sci ) 30 min at RT and then overnight at 4°C. Secondary peroxidase-conjugated antibodies (Dako, Glostrup, Denmark,goat-anti-mouse (for SOD1), goat-anti-rabbit (for eNOS, CAT), mouse-anti-goat (for nNOS)) were diluted 1:200 and applied in blocking buffer for 1 hour at RT. Staining was visualized with 3,3’-diaminobenzidine (DAB, Dako), sections were counterstained for 2 min with Mayer’s Hematoxylin (Sigma-Aldrich), and imaged using an Olympus BX51 microscope equipped with a DP26 camera and CellSens software (Olympus). Presence of immunohistochemical staining was semi-quantitively analyzed by rating the stainings (0 = no staining; 4 = strong staining). Rating was performed by two independent observers that were blinded to the sections’ IDs. Sections exposed to the secondary but not the primary antibody were used as negative controls. Sections of human kidney, thoracic aortic aneurysm, atrial appendage, or parietal pericardium, formalin-fixed directly after isolation during surgery, served as positive controls.