Carotid Plaque Histological Analysis

Carotid plaques were removed by standard surgical techniques and minimal manipulation to the specimens. Immediately after the surgery, plaques were stored in phosphate buffered saline (PBS) at 4°C. The specimens were embedded into an optimum cutting temperature (OCT) compound (LEICA, 020108926) and stored at -80°C for further analysis. Samples were analyzed by immunohistology, immunohistochemistry, and immunofluorescence using confocal microscopy. For immunohistochemistry plaque samples were analyzed for various CD cellular markers including CD66b, CD163, CD68, and lipids. Additional cellular markers were used for confocal microscopy. Quantitative analyses of the expression of various markers were performed as previously described (2 (link), 15 (link)) and as specified below. Mouse monoclonal primary antibodies were used to identify neutrophils (anti-CD66b), macrophages-foam cells (anti-CD163) and anti-3-nitrotyrosine for oxidative-nitrosative stress. Rabbit primary polyclonal antibodies were used for double-labeling the cells with additional markers including scavenger receptors anti-CD68 and anti-CD36, anti-NE, anti-MPO, anti-Vascular Endothelial Growth Factor (VEGF), anti- CD31, and anti- smooth muscle actin (SMC-actin). Polyclonal anti-CD163 was used for double-labeling with anti-CD66b. Intra/extra cellular lipids and lipid crystals were determined by immunohistochemistry with Oil Red O staining (15 (link)).