Necropsy, bronchoalveolar lavage (BAL) and lung tissue collection:

Mice were euthanized 5 weeks after HCT using CO₂ and their lungs were surgically exposed. The trachea was cannulated and lungs were lavaged with 5 aliquots of 800 μL of 0.9% saline (VWR International, West Chester, PA). BAL fluid supernatant from the first 2 lavage aliquots was stored at −80°C until further use. BAL cells underwent red blood cell lysis and were subsequently counted using a hemocytometer. Lungs were perfused with 0.9% saline. The right lung was snap-frozen in liquid nitrogen and stored at −80°C for subsequent collagen (hydroxyproline) analysis. The left lung was gravity-inflated with 10% formalin (VWR International, West Chester, PA), fixed in 10% formalin solution for 24 hours and then transferred into 70% EtOH. The entire left lung was subsequently embedded in paraffin, three to four 5 μm sagittal sections were placed on each slide and stained with hematoxylin and eosin (H&E) and Masson-Trichrome stains. Histologic analysis for each sample was based on three sections spanning the entire left lung.