The middle lobe of the left lung was cut off and fixed by 4% paraformaldehyde, for preparation to be embedded by paraffin, and then routinely processed. Lung tissue sections were stained with hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Masson's trichrome and then measured with the Nikon ECLIPSE 80i biomicroscope and NIS-Elements BR 3.2 image analysis system (Nikon, Japanese) according to our previous study [38 (link)].
We surveyed the perimeter of basement membrane (Pbm), total area of bronchus (Wat1), area of lumen (Wat2), area of outer margin of the smooth muscle (Wam1), and area of medial smooth muscle (Wam2) in H&E-stained sections; then calculated the standardized thickness of airway wall (Wat, Wat = (Wat1 − Wat2)/Pbm) and the standardized thickness of airway smooth muscle (Wam, Wam = (Wam1 − Wam2)/Pbm); observed the goblet cells and Pbm in PAS-stained sections; then calculated the standardized number (number/Pbm) and area (area/Pbm) of PAS-positive goblet cells; determined collagen fiber area (stained in blue) and Pbm in Masson's trichrome-stained lung sections; and then calculated the mean score of the fibrotic area divided by Pbm in each rat.
We surveyed the perimeter of basement membrane (Pbm), total area of bronchus (Wat1), area of lumen (Wat2), area of outer margin of the smooth muscle (Wam1), and area of medial smooth muscle (Wam2) in H&E-stained sections; then calculated the standardized thickness of airway wall (Wat, Wat = (Wat1 − Wat2)/Pbm) and the standardized thickness of airway smooth muscle (Wam, Wam = (Wam1 − Wam2)/Pbm); observed the goblet cells and Pbm in PAS-stained sections; then calculated the standardized number (number/Pbm) and area (area/Pbm) of PAS-positive goblet cells; determined collagen fiber area (stained in blue) and Pbm in Masson's trichrome-stained lung sections; and then calculated the mean score of the fibrotic area divided by Pbm in each rat.
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