Quantifying Aortic MMP-2 and MMP-9 Localization

In order to assess MMP-2 and MMP-9 localization in the thoracic aorta, the de-waxed aortic sections were analyzed using specific antibodies (ab37150; Abcam and ab19016; Millipore, respectively), and an immunohistochemical technique using the R.T.U. Vectastain ABC kit (Vector Laboratories, Inc., CA, USA) as previously described [40] (link). The experimental conditions were optimized to generate a strong and specific signal for the antigen of interest. The measurements were performed in a single batch and the specimens were stained together with a positive control at the same time. The stained sections was examined with light microscopy and the images were captured at ×400. The amount of MMP2 or MMP-9 in the aortic wall was measured, as were collagen and elastin, by counting the thresholded pixels stained with antibodies to MMP-2 or MMP-9 using the Image-Pro Plus Program (Media Cybernetics, MD, USA). All measurements were made by one observer and preliminary observations of intra-observer repeatability showed a coefficient of variation of less than 5% for all estimations of thresholded area.