Blood, BALF, and Tissue Collection

Mice were euthanized via intraperitoneal injection with sodium pentobarbital at 56 mg/kg body weight. Using heparinized syringes, blood was collected from the abdominal aorta and centrifuged at 3500 x g for 10 min at 4°C to separate the erythrocytes from plasma, and then stored at -80°C. Plasma was later analyzed for blood urea nitrogen (BUN) levels using a colorimetric detection kit (Thermo Fisher Scientific, Wilmington, DE). Bronchoalveolar lavage fluid (BALF) was collected from the lung as described previously (36 (link)). The left lung lobe was fixed in 10% neutral buffered formalin (NBF) (Fisher Scientific, Pittsburgh, PA) at a constant pressure of 30 cm H₂O for a minimum of 1 h, then immersed in a large volume of the fixative until further routine processing for light microscopic examination and morphometric analyses described below. Spleens were weighed, then half was stored in NBF along with one kidney until processed for histology. The caudal lung lobe and half a kidney were stored individually in RNAlater (Thermo Fisher Scientific, Wilmington, DE) at 4°C. The remainder of the right lung, half kidney, and half spleen were snap-frozen in liquid nitrogen and stored at -80°C.