Mean linear intercept (MLI) analysis

For alveolar morphometric analysis, mouse lungs were processed according to the above protocol for paraffin embedded samples, with the exception of inflation with 4% PFA at a constant pressure of 25 cm H₂O. The paraffin-embedded lung sections were stained by hematoxylin and eosin (H&E) for analyzing alveolar morphology metric. At least 5 randomly selected sections from each genotype were selected for analysis. The mean linear intercept (MLI) was calculated as the linear sum of the lengths of all lines randomly drawn across the images, divided by the number of intersections between alveolar walls. A minimum of 1000 intercepts from 60 lines drawn across the lung in a randomized fashion were obtained for each lung, and the analysis was carried out on Fiji with the “Cell Counter” plug-in. The space of airway and pulmonary vessels was excluded. Statistical analysis was done using the one tailed Student’s t-test. Data are presented as means ± SD.