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. 2005 Nov;167(5):1349–1359. doi: 10.1016/S0002-9440(10)61222-9

Figure 4.

Figure 4

Location and time course of HU177 cryptic collagen epitope exposure after onset of hindlimb ischemia. A: Frozen sections (×40) of ischemic muscle harvested at days 0 (pre-op), 1, 3, 7, 14, and 30 days were stained for the HU177 cryptic collagen epitope using mAb QH2B. Sections were counterstained with horseradish peroxidase for detection of antibody. B: Frozen sections (×40) of ischemic muscle harvested at day 7 were costained for the cryptic collagen epitope, HU177, and the endothelial cell marker, CD31. HU177 is denoted with the brown horseradish peroxidase staining, whereas CD31 is indicated by the red alkaline phosphatase staining. The solid arrows indicate CD31 staining of blood vessel endothelial cells in the ECM between ischemic skeletal muscle cells. The arrowheads indicate HU177 staining in the ECM, which colocalizes with and surrounds CD31+ endothelial cells. C: Quantification of HU177 staining was performed using digital images of stained sections. Sections were analyzed by blinded observers using Adobe Photoshop. The number of mAb QH2B-stained pixels in each image was determined and expressed as a ratio of the total number of pixels of muscle tissue present in each slide. Each time point is the average value obtained from analyzing three to five representative microscopic fields from muscle sections from animals at each time point (n = 3) for HU177 staining. HU177 exposure parallels increased MMP-9 and MMP-2 activity and limb reperfusion. The asterisk denotes a significant difference (P < 0.05) between nonischemic tissue at day 0 (preoperative) and ischemic tissue at days 7 and 30.