Lungs were pressure-fixed
in situ via instillation with 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) at 20 cm (H
2O) via a cannula inserted into the trachea [
17]. PFA-saturated lungs were then excised with rib-cages and post-fixed for 24 h in 4% PFA at 4°C. The following day, lungs were removed and placed into Zamboni’s fixative for 24 h at room temperature (RT) [
18]. Lungs were embedded in paraffin and serially sectioned as 5–6 μm serial coronal sections in the ventral to dorsal plane. Slides were randomly picked from each group for staining and analysis. Sections on glass slides were deparaffinized by immersion in xylene (3 × 5 min), 100% ethanol (3 × 5 min) and tap water (1 min). Lung sections were stained for histological analyses using four methods: hematoxylin and eosin stain (Mayer’s hematoxylin – 3 min, acid alcohol – 3 seconds (s), Scott’s tap water – 10 s, eosin – 3 min, rinsing in tap water between solutions), Masson’s trichrome stain (Celestin blue – 5 min, Mayer’s hematoxylin – 3 min, acid alcohol – 3 s, Biebrich scarlet/acid fuchsin – 5 min, 1% phosphomolybdic acid – 5 min, aniline blue – 5 min, rinsing in tap water between solutions), periodic acid and Schiff’s (PAS) stain (periodic acid – 5 min, Schiff’s reagent – 5 min, hematoxylin – 5 min, acid alcohol – 3 s, Scott’s tap water – 10 s, rinsing in tap water between solutions) and modified Weighert’s elastin stain (0.25% potassium permanganate – 5 min, 5% oxalic acid – 3 s, resorcin-fuchsin – overnight, tartazine in saturated picric acid – 3 min, rinsing in tap water between solutions) [
18,
19]. All stains were prepared according to standard protocols [
19]. After staining, slides were dehydrated in 100% ethanol and xylene (3 × 5 min) and mounted in DPX mounting medium (BDH Laboratory Supplies, #360294H) [
19]. Five to seven photographs were taken per section (depending on lung size) using a light microscope (Olympus CKX41) and digital camera to assess the entire alveolar area [
15]. Photographs of lung sections were analyzed for the following characteristics: mean linear intercept (L
m), tissue area, alveolar number, elastin-positive blood vessel number per tissue area, secondary septum (crest) number per tissue area, elastin-positive tissue per total tissue area, the presence of mucus and mucin-secreting cells, the presence of collagen depositions and fibrosis. To evaluate the number of alveoli, three vertical and two horizontal lines were drawn randomly across the lung section image. The total lengths of vertical and horizontal lines were equal. The summary length of all lines per image was 3.63 mm. The average number of alveoli was calculated from the number of airspaces (including alveolar ducts, but excluding airways) appearing on those lines [
15]. Mean linear intercept (L
m) was calculated from measurements of the lengths of airspaces (both alveoli and alveolar ducts, but excluding airways) falling on top of these lines. The number of vertical measurements was equal to the number of horizontal measurements. Secondary septa and blood vessels were detected by the presence of elastin that was stained black by resorcin-fuchsin (Weighert’s elastin stain) [
20,
21]. The number of secondary septa and the amount of elastin were calculated in relation to the area of tissue per total area of image. The number of blood vessels was calculated both per total image area and per tissue area. The tissue area was calculated using Image-Pro Plus software (Media Cybernetics) by selecting all colored pixels on the section image and then transferring them into microns according to the scale bar. The amount of elastin was calculated by determining the total percentage of elastin-positive tissue using Image-Pro Plus.