B25380

The femoral tissues were collected and paraffin-embedded (S25190, Yuanye), fixed on a microtome (RM2235, Leica, Solms, Germany) and sliced into 8 μm thick. Then, the slices were fixed on a glass slide (80,302–3101-16-P4, ShiTai, Jiangsu, China) and deparaffinized. Next, the tissue slices were incubated with hematoxylin (B25380, Yuanye) for 10 mins and then with eosin (G1100, Solarbio, Beijing, China) for 1min at room temperature. Finally, indexes were detected using a phase-contrast optical microscope (Magnification ×100 and ×200) (Axio Lab.A1 pol; Leica, Solms, Germany).

Lung and airway tissues were fixed with 4% paraformaldehyde (R008069, Rhawn) for 24 h at room temperature. Then the samples were washed overnight with tap water and dehydrated with gradient ethanol (E809057, Macklin, Shanghai, China) and double distilled water. Subsequently, the tissues were embedded into paraffin, sectioned, and dewaxed. Then the tissues or cells in BALF were stained by hematoxylin (B25380, Yuanye, Shanghai, China) for 10 min and further incubated with hydrochloric acid alcohol (AR0038, Boster, Wuhan, China, http://www.boster.com.cn/index/index.html) for 5 min. Then the samples rested in 50°C warm bath until the tissues appeared blue, and were further stained with eosin (C0109, Beyotime) for 1 min at room temperature. After soaking the samples in gradient ethanol again for dehydration, the sample slices were made transparent by soaking in xylene (R017750, Rhawn). Finally, the sample slices were sealed with neutral gum (N116470, Aladdin, Shanghai, China) and the image of each section was collected under a DMLA full automatic microscope (Leica, Solms, Germany) at a magnification of 100 ×.