Cx41 light microscope

The tracheal tissues of the normal and model groups were fixed with 4% paraformaldehyde solution at 25°C for 24 h, then rinsed for 6–8 h and dehydrated with ethanol solutions of 70, 80 and 90% and a mixture of pure alcohol and xylene at 25°C for 15 min, xylene I for 15 min and xylene II for 15 min until tissues were transparent. Tissues were incubated in a mixture of xylene and paraffin at 25°C for 15 min and then placed in paraffin I followed by paraffin II at 25°C for 50–60 min each. Tissues were paraffin-embedded and sliced (5 µm thick). The paraffinized sections were baked at 45°C and Paraffin sections were dewaxed with xylene I followed by xylene II for 5 min each, then placed in 100, 95, 90, 80 and 70% alcohol solutions for 3 min each, and then placed in distilled water for 3 min. Once tissues were washed with distilled water, each section was placed in an 0.2% aqueous solution of hematoxylin at 25°C for 3 min and incubated in an 1% ethanol differentiation solution at 25°C for 15 sec, washed slightly with distilled water and returned to the blue hematoxylin solution at 25°C for 15 sec. Sections were then rinsed with water, stained with 0.5% eosin at 25°C for 3 min and rinsed with water again. Sections were placed in 95 and 100% alcohol for 3 min each for dehydration until transparent, sealed and examined by an Olympus CX41 light microscope at ×400 magnification.

All samples blocked with FSC were cut into 8 µm-thick sections using the LEICA CM1950 (LEICA, Wetzlar, Germany), and sections were stained with hematoxylin-eosin (HE) staining. Microscopic observation was carried out with an Olympus CX41 light microscope (Olympus Co., Tokyo, Japan). The muscle cross-sectional area (CSA) was quantified using ImageJ software (http://rsb.info.nih.gov/ij/index.html, 11 June 2019).