Evaluation of angiogenesis of the ischemic model mice. (A-C) Typical images of the gastrocnemius immunostained with an anti-CD31 antibody. The muscles from ischemic legs were isolated at 7, 14, and 28 days after the induction of ischemia and immunostained. Brown circles and dots represent CD31⁺ capillaries. Scale bar represents 50 μm. (D) Measurement of CD31⁺ capillary density. In the gastrocnemius of the ischemic or control leg, capillaries stained with the anti-CD31 antibody were counted. Solid and dotted lines represent the sample derived from ischemic and control legs, respectively. n = 4. Error bars represent SEM. (E-J) Changes in blood perfusion were assessed using laser Doppler perfusion imaging in ischemic legs of model mice. (E-F) Images before and soon after operation. After operation, the perfusion signal in the ischemic leg is significantly decreased. (G-J) Images from 7, 14, 21, and 28 days after operation. Blood perfusion in an ischemic leg gradually increases. (K) Change in perfusion units as determined by this imaging technique. Relative ratios of mean perfusion units between ischemic and control legs in the same mouse are shown (ischemic leg value/control leg value). n = 6. Error bars represent SEM.