Emmprin

Streptavidin-Peroxidase kit (Zhongshan, Beijing, China) was used according to the instructions. Polyclonal antibody EMMPRIN and MMP-9 (dilution 1:150, Abcam, UK) were applied. Osteocalcin (OCN) and osteopontin (OPN) (dilution 1:100, Bioss, China) were performed to determine the expression of these proteins in the alveolar bone healing. PBS was obtained as negative control.

The aorta (from the arch to abdominal aorta) was used. Serial 6 μm sections (40 sections per mouse) of the aorta were collected for immunohistochemical staining and Masson’s trichrome staining. Immunohistochemistry was used to characterize macrophages (CD68, dilution 1:80, Abcam, Cambridge, UK); α-smooth muscle actin (α-SMA, dilution 1:100, Abcam); EMMPRIN (dilution 1:250, Abcam); MMP-9 (dilution 1:300, Abcam); VCAM-1 (dilution 1:200, Abcam); ICAM-1 (dilution 1:150, Abcam); and NFκB p65 (dilution 1:50, Cell Signaling Technology, Inc.) as previously described [13 (link)]. Immunohistochemical staining was visualized using an EnVision kit (DAKO) according to the manufacturer’s instructions. Collagen content in the tissue was assessed by Masson’s trichrome staining, similar to the methods previously described [19 (link)]. Images were captured with a light microscope camera system (Olympus, Tokyo, Japan) and analyzed using Quantity One analysis system. The results were expressed as the percentage of stained area to total plaque area [20 (link),21 (link)]. Fibrous cap thickness of lesion area was evaluated on the most advanced lesion in each mouse by using α-SMA-stained slides.