Eclipse 80i biomicroscope

The tibia was fixed in a 10% formaldehyde solution and then used to make a 5 μm thick longitudinal section. The antigen of the sections was repaired with citrate buffer at pH 6.0 after gradient dewaxing and then blocked with goat serum. The sections were incubated with primary antibodies against the receptor activator of nuclear factor-kappa B ligand (RANKL), the macrophage colony-stimulating factor (M-CSF), Wnt5a, the d2 isoform of vacuolar (H⁺) ATPase (v-ATPase) V0 domain (Atp6v0d2), osteoprotegerin (OPG), insulin-like growth factor-1 (IGF-1), transforming growth factor-β1 (TGF-β1), and bone morphogenetic protein-2 (Bmp-2) (1:500), respectively, at 4°C overnight. Subsequently, the sections were washed with PBS and incubated with corresponding secondary antibody (1:200, Zhong Shan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h. The sections were then costained with DAPI and then observed. We used a Nikon ECLIPSE 80i biomicroscope to magnify 200 × and selected 3 random regions for each section. IOD were measured using NIS-Elements BR 3.2 image analysis system (Nikon, Japan). Then calculate the average of the three regions as the final result of the sample.

The right lungs were lavaged 3 times with 3 mL, 3 mL and 4 mL ice-cold saline using a tracheal cannula and a 5 mL polyethylene syringe. The cell-debris pellets of bronchoalveolar lavage fluid (BALF) samples were collected after centrifugation (500 rpm, 5 min, and 4 °C). A differential cell count was performed on cytospin by Wright-Giemsa staining with the Nikon ECLIPSE 80i biomicroscope and NIS-Elements BR 3.2 image analysis system (Nikon, Japanese). The number of lymphocytes and eosinophils in 200 cells was counted.

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.