Paraffin-embedded carotid arteries were de-paraffinized in xylene, rehydrated in graded ethanol, and subjected to immunostaining. Immunohistochemistry was performed on serial sections, as described previously [12 (link)]. The primary antibodies used were CD74 (Sigma, St Louis, MO, USA), CD68 clone PG-M1 (Dako, Denmark), smooth muscle actin clone 1A4 (Dako, Denmark), ferritin (Dako, Denmark), and thrombin receptor (protease-activated receptor 1, Sigma). The immunoreactions were visualized using the EnVision+/horseradish peroxidase (Dako, Denmark) method and ChemMate EnVision Detection Kit (Dako, Denmark). Control sections without primary antibodies or with non-immune IgG were run for each protocol, resulting in consistently negative results. The slides were counterstained with haematoxylin.
All histological sections were examined under a light microscope, and the images were digitalized with Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5, Adobe Systems Incorporated, San Jose, CA, USA) as described previously [12 (link)]. The individual responsible for analysis was blinded to patient information.
All histological sections were examined under a light microscope, and the images were digitalized with Image Grabber program (Toronto, ON, Canada). The microscope was set on the same parameters used to scan all samples. The randomly digitalized images were analyzed with Adobe Photoshop (v5.5, Adobe Systems Incorporated, San Jose, CA, USA) as described previously [12 (link)]. The individual responsible for analysis was blinded to patient information.