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.