The mouse heart was pulled toward the liver to expose the pulmonary artery (PA) and pinned down through the heart apex with a 1 ml empty syringe connected with a needle. A silk suture was wrapped loosely around the PA as close to the heart as possible. A small incision was made in the right ventricle with spring scissors. Tubing (PE50, BD bioscience, cat #427411) ~15 cm long (stretched at the tip) connected to (23G) needle was inserted into the PA through the incision in the right ventricle and tightened with the suture. A total of 1 ml of DPBS was injected into the lung via PA to remove all the remaining blood. Afterwards, to expand the collapsed lung, the trachea was exposed and cleaned from surrounding tissue. A silk suture was loosely wrapped around the trachea and a small incision was made into the trachea with spring scissors. Tubing (PE50) ~15 cm long connected to (23G) needle was inserted into the trachea and tightened with the suture. One ml of DPBS was injected into the lungs via trachea – this inflates the collapsed lungs. A 1 ml syringe filled with yellow MICROFIL was connected to the tubing inserted into the PA, and MICROFIL was injected into the PA vasculature until all the blood vessels were filled. Massaging the lung with a cotton swab while injecting helped to disperse the MICROFIL. After the vasculature was completely filled, the tubing was removed and the suture around the PA tightened to prevent MICROFIL leakage. For airways injection, a 1 ml syringe filled with green MICROFIL was connected to the tubing inserted into the trachea, and MICROFIL was injected into the trachea until the lung was entirely filled with MICROFIL. Massaging the lung with cotton swab while injecting again helped to disperse the MICROFIL. After the lung was completely filled, the tubing was removed and the suture around the trachea tightened to prevent MICROFIL leakage.

Lungs were dissected out and placed at 4°C ON to allow the MICROFIL to solidify. The next day the lung was fixed with 3.7% FA diluted in DPBS. After 24 hr, lungs were washed and kept in DPBS. Lungs were separated into lobes and the right superior lobe was used for µCT scanning.

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