To examine the homing and incorporation of EPCs in the ischemic muscle, ischemic gastrocnemius muscle and/or soleus muscle were embedded in OCT medium for frozen section. The GFP positive EPCs were counted in randomly selected fields for a total of 20 different fields (×40 magnification) per section and 3 sections per animal. The EPC incorporation was expressed as the percentage of GFP positive capillaries.
The extent of angiogenesis and arteriogenesis at day 28 post ischemic surgery was assessed by measuring capillary density in gastrocnemius muscle and/or soleus muscle using isolectin B4 staining, and arteriole area in adductor muscle. Ischemic gastrocnemius muscle, soleus muscle and/or adductor tissues were fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections were cut at 5 μm and stained with Alexa Fluor® 594 conjugated isolectin GS-IB4 (Thermo Scientific, Waltham, MA)or anti-alpha smooth muscle actin (α-SMA) antibody (Abcam) to evaluate the capillary density and arteriole area respectively. The number of capillaries or arteriole area were calculated in randomly selected fields for a total of 20 different fields (×40 magnification) per section and 3 sections per animal. The capillary density was expressed as capillary number per muscle fiber, and the arteriole area was expressed as α-SMA positive arteriole area per fiber and normalized by control.
The extent of angiogenesis and arteriogenesis at day 28 post ischemic surgery was assessed by measuring capillary density in gastrocnemius muscle and/or soleus muscle using isolectin B4 staining, and arteriole area in adductor muscle. Ischemic gastrocnemius muscle, soleus muscle and/or adductor tissues were fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections were cut at 5 μm and stained with Alexa Fluor® 594 conjugated isolectin GS-IB4 (Thermo Scientific, Waltham, MA)or anti-alpha smooth muscle actin (α-SMA) antibody (Abcam) to evaluate the capillary density and arteriole area respectively. The number of capillaries or arteriole area were calculated in randomly selected fields for a total of 20 different fields (×40 magnification) per section and 3 sections per animal. The capillary density was expressed as capillary number per muscle fiber, and the arteriole area was expressed as α-SMA positive arteriole area per fiber and normalized by control.