Tissues were dissected from adult mice that were anesthetized with ketamine/xylazine and PBS-perfused via the aorta. After overnight fixation at 4 °C with 4% paraformaldehyde in PBS, tissues were washed with PBS, and cryoprotected in 20% sucrose. Tissues were then infiltrated overnight at 4 °C and embedded the next day with the same 2:1 mixture of 20% sucrose and O.C.T. compound before being frozen on dry ice.
For identification of vascular cell type, tissue sections were subjected to immunoperoxidase staining. After washing with PBS and permeabilization with PBS containing 0.2% Tween 20 (PBST), endogenous peroxidase activity was blocked by incubation with 1% H2O2 in PBS for 10 min. Non-specific binding was blocked by incubation with blocking buffer (3% rabbit serum in PBST) for 30 min at room temperature. Tissue sections were incubated with either rat anti-mouse CD31 (1:250, BD Pharmingen) or rat anti-mouse EMCN (1:1,000, mAb V.7C7, Santa Cruz) diluted in blocking buffer overnight at 4 °C. A section incubated with rat IgG as a negative control was included in each experiment. Slides were examined and images were captured using a Zeiss Axioskop 2 MOT microscope with a mounted AxioCam digital camera under both × 20 and × 40 objective lenses. Post-acquisition processing of images was conducted using Adobe Photoshop.
For identification of vascular cell type, tissue sections were subjected to immunoperoxidase staining. After washing with PBS and permeabilization with PBS containing 0.2% Tween 20 (PBST), endogenous peroxidase activity was blocked by incubation with 1% H2O2 in PBS for 10 min. Non-specific binding was blocked by incubation with blocking buffer (3% rabbit serum in PBST) for 30 min at room temperature. Tissue sections were incubated with either rat anti-mouse CD31 (1:250, BD Pharmingen) or rat anti-mouse EMCN (1:1,000, mAb V.7C7, Santa Cruz) diluted in blocking buffer overnight at 4 °C. A section incubated with rat IgG as a negative control was included in each experiment. Slides were examined and images were captured using a Zeiss Axioskop 2 MOT microscope with a mounted AxioCam digital camera under both × 20 and × 40 objective lenses. Post-acquisition processing of images was conducted using Adobe Photoshop.