MICROFIL (Flow Tech Inc) was prepared as follows. Yellow MICROFIL (Y) cat. #MV-122 was diluted with clear MICROFIL (C) cat. # MV-Diluent in 3:1 (Y:C). Blue MICROFIL (B) cat. #MV-120 was diluted 1:1 (B:C) with clear MICROFIL. Diluted yellow MICROFIL was mixed with diluted blue MICROFIL 1:1 creating a green MICROFIL. Yellow MICROFIL was injected into common bile duct (CBD) or pulmonary artery (PA). Green MICROFIL was injected into portal vein (PV) or trachea (TA). 1 ml of diluted MICROFIL is mixed with 50 μl of hardener (supplied by Flow Tech Inc) prior injection.

Postnatal day 15 (P15) mice were sacrificed by decapitation and perfused through the heart with 3 ml of Hanks' Balanced Salt solution (HBSS) (Life Technologies cat. # 14025092). Adult mice were sacrificed by CO2 inhalation and perfused through the heart with HBSS for 3 min (perfusion rate 5 ml / 1 min). For liver resin injections, the mice were perfused trough the left ventricle, for lung resin injections the mice were perfused though the right ventricle.

A small transversal incision was made in inferior vena cava with spring scissor to release the liver vascular pressure. CBD was exposed by moving aside the liver and intestine and cleaned from surrounding tissue in area about 5 mm long. Silk suture (Agnthos AB cat. #14757) was loosely wrapped around the cleaned CBD. A longitudinal CBD incision was made at the spot where CBD enters the pancreas next to sphincter of Oddi by spring scissor. The tubing (PE10, BD Biosciences cat. # 427401) ~15 cm long was prepared by stretching one side of the tube until the diameter becomes thin enough to fit into CBD. Diagonal cut is made at the tip of the tubing while the other side contains needle (27G) connected to the syringe filled with MICROFIL. The tubing connected to a syringe and filled with a yellow MICROFIL was inserted into the CBD, the suture around the CBD can be tighten to secure the tubing in place. Yellow MICROFIL was injected into the CBD until resistance was met or MICROFIL spots were visible on the liver surface. Massaging the liver with cotton swab while injecting helped to disperse the MICROFIL. The tubing was removed and silk suture was tightened around the CBD to prevent leakage.

PV was cleaned from surrounded tissue. A small incision was made in PV using spring scissor. Silk suture was loosely wrapped around the cleaned PV above the incision. Tubing (PE10) ~15 cm long connected to (27G) needle was inserted into the PV incision and secured with silk suture. Green MICROFIL was injected into the PV until blood vessels on the surface were filled or resistance was met. Massaging the liver with a cotton swab while injecting helped to introduce the MICROFIL. The tubing was removed and silk suture was tightened around the PV to prevent leakage.

Liver was dissected out and placed at 4°C overnight (ON) for MICROFIL to solidify. The next day the liver was fixed with 3.7% formaldehyde solution (FA) (Sigma-Aldrich cat. #F1635) diluted in Dulbecco's phosphate-buffered saline (DPBS) (Life Technologies cat. # 14190144). After 24 hr, liver was washed and kept in DPBS. Liver was separated into lobes. The left lateral lobe was placed in 50% methanol (Sigma-Aldrich, cat. # 322415) for 4 hr and into 100% methanol ON. Further, the lobe was placed in benzyl alcohol (Sigma-Aldrich, cat. #402834) and benzyl benzoate (Sigma-Aldrich, cat. #B6630) (BA:BB 1:2) solution until transparent. The right medial lobe (only FA fixed) was used for µCT scanning. Liver lobe images of right medial lobe (P15) and left lateral lobe (P15 and adult) were taken using a stereomicroscope Stemi 305 (Carl Zeiss Microscopy) with a PowerShot S3 IS camera (Canon) or iPhone6 connected to a LabCam adapter.

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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