2.5. Immunohistochemical analysis
Immunohistochemical analysis of MMP-9 expression was performed in the central section of the plaque and one of the adjacent sections. Primary antibodies used in the study were: MMP-9 (Leica Mikrosysteme Vertieb GMBH, UK, monoclonal, clone 15W2, 1:50 dilution), CD68 PG-M1 (DakoCytomation, Denmark, monoclonal, clone PG-M1, RTU), smooth mucle actin (SMA) (DakoCytomation, Denmark, monoclonal, clone 1A4, RTU), CD34 (DakoCytomation, Denmark, monoclonal, clone QBend 10, RTU).
Specimen sections were deparaffinized and antigens MMP-9, CD68 PG-M1, CD34 and SMA were unmasked in PT-module (DakoCytomation, Denmark) at 97⁰C, 20 minutes in buffer derived from EnVision flex commercial ”3 in 1” set (K8010, DakoCytomation, Denmark), and were then processed using a standardised automated procedure in an Autostainer device (DakoCytomation, Denmark), and stained with haematoxylin. Specimen sections were used as positive controls for the investigated biomarkers according to the manufacturer’s recommendations. Semiquantitative analysis of macrophages and SMA by staining with CD68 PG-M1 antibody and SMA antibody, respectively, was performed according to the study byVerhoeven et al. [30]. Histological analysis of a type of bleeding was performed by determining the average density of small blood vessels by immunohistochemical staining of CD34 as described in the study by Derksen et al. [32]. The expression of MMP-9 protein was semiquantitatively analysed on specimen sections in macrophages and VSMC: 0=negative, 1=rare cells or one cluster of ≥20 cells, 2=less than 5 clusters of ≥20 cells, 3= ≥5 clusters consisted of ≥20 cells or one cluster ≥100 cells [33]. Analysis of endothelial cells was performed over 10 large areas with high magnification up to 400x, and the results were demonstrated as: 0=negative, 1= <25%, 2= 25-50%, 3= >50-75% and 4= >75% of stained endothelial cells. The intensity of MMP-9 expression was demonstrated as: 0=negative, 1=visible using 400x magnification, 2=100x magnification and 3=40x magnification. Semiquantitative analysis of plaque specimen, immunohistochemical staining of macrophages, VSMC, microvascular density (MVD) and MMP-9 protein was performed using the software ”ToupTekView” (Copyright ©, version x64 3.7.8481, www.touptek.com). Immunohistochemical examinations were performed independently by two experienced investigators blinded to the clinical history and MRI findings. Any discrepancies were resolved by discussion between the two investigators.