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Following ex vivo autoradiography and staining with ORO, en face aortic arches were embedded in paraffin wax (TissuePrep), sliced into 5 µm sections, and deparaffinized. Regions which were positive during ex vivo [ 18 F]FMBP autoradiography were selected for sectioning (n = 2 per aorta). Antigen retrieval was performed in citrate buffer (pH 6.0) at 100 °C for 150 seconds.
Sections were covered in 10% normal goat serum (Vector Laboratories) for 10 minutes and incubated with 1:100 MMP-13 or MMP-2 primary antibody (ab39012 or ab97779, Abcam) and 1:500 Mac-2 primary antibody (CL8942AP, Cedarlane) for 16 hours at 4 °C. Samples were then incubated with secondary antibodies at 1:500 dilution for 30 minutes (MMP-13 & MMP-2: A-11037, Mac-2: A-11006, Invitrogen). Nuclei were counterstained with Hoeschst 33258 (10 mg/mL in PBS) for 8 minutes. Slides were coated with fluorescent protective mounting media (Dako), dried, and covered until imaging by fluorescent microscopy (Zeiss Axio Imager A2). Isotype control antibodies (rabbit IgG ab171870, Abcam and rat IgG CLCR2A00, Cedarlane) were also utilized to assess non-specific fluorescence at equivalent concentrations to the primary antibodies.
Images were acquired with Aperio ImageScope (10× magnification), stitched using Microsoft Image Composite Editor, cropped using Adobe Photoshop CS5 and quantified by 2 independent observers using ImageJ.