Lung histology and morphometry

For quantification of alveolarization, lungs were infused with 2% paraformaldehyde via a tracheal catheter at a uniform pressure of 25 cm H₂O for 3 min using a 25G or 21G catheter for 2-week old and 2-month old rats respectively. Hematoxylin and eosin-stained tissue sections were used to obtain 10 random pictures of each lung at ×20 magnification. The images were viewed under a field of equally spaced horizontal lines using ImageJ and mean linear intercept (MLI) was calculated as the average of total length of lines divided by the total intercepts of alveolar septa from each lung. Image analysis was performed by a blinded observer.