1. Anesthetize mouse with intraperitoneal (i.p.) injection of pentobarbital (120 mg/kg body weight). Make sure that the mouse is deeply anesthetized (i.e., no reflex response while pinching lower limbs).

2. Restrain mouse on surgical pad under microscope with ventral side facing up.

3. Make a 2- to 3-cm horizontal incision in the abdomen just below the diaphragm, cut ribs vertically through the diaphragm to open the chest and expose the heart and lungs.

4. Heparin was injected in the right ventricle to prevent blood coagulation.

5. Remove the adipose tissue and thymus around the heart and large vessels (aorta and main PA).

6. Prepare a catheter using PE-20 tubing of 10-cm length attached with 25G needle.

7. Make a small incision at the base of the main PA and insert the catheter into PA via the right ventricle and perfuse warm PBS at a speed of 0.05 ml/min for 3 min using an automated syringe pump (Micro-Harvard) until lung turns completely white.

8. Freshly prepare the Microfil polymer casting solution and infuse 0.08 ml of Microfil into the lung via the RV and PA until lung appears yellow. Make sure that Microfil polymer mixture has no air bubbles.

9. Cover the Microfil polymer-filled lungs (and heart) with saline-soaked paper towel to avoid desiccation and let it set overnight at 4°C.

10. Next day, after Microfil is polymerized, isolate the heart and lungs, place them in 15 ml polypropylene centrifuge tube filled with PBS, and place the tubes on a gentle shaker at room temperature for 15 min.

11. Replace PBS in the tubes with 50% ethanol and gently shake the tube for 1 h at room temperature. Replace 50% ethanol with 70% ethanol and gently shake the tube for 1 h; repeat this step for 80%, 95%, and 100% (2 times for 100% ethanol), respectively, to completely dehydrate the lung tissue.

12. Replace 100% ethanol in the tube with methyl salicylate (under a fume hood) and gently shake the tube overnight at RT until the lung tissue becomes translucent with Microfil clearly visible.

13. Take photograph of the lungs with a microscope camera at 8× and 30× magnification.

14. Use Adobe Photoshop software to manually outline the lung peripheral region (1 mm width from the edge of the lung) for vascular density analysis.

15. Use NIH Image J software to convert the manually outlined image to the binary image, and calculate the total length of vascular branches, the number of vascular branches and the number of junctions in the skeletonized images. Data were normalized by the area selected within the edge of peripheral vascular lung.