TUNEL staining assay

The apoptotic cells in rat lung were identified by TUNEL staining (Roche Applied Science). Briefly, deparaffinized and fixed sections were immersed in 20 mg/ml proteinase K for 15 min. After refixation and equilibration, sections were incubated with 50 μl of TUNEL reaction mixture at 37°C for 1 h. Sections were also stained with Hoechst. Images were viewed under an inverted microscope (Leica DMI3000B, Wetzlar, Germany). The airways of 2 mm and less in diameter were pictured. For apoptotic cells, we examined positive fluorescent apoptotic nuclei in 20 low power fields/slide at 10 × magnification 40 fields per section were randomly manually selected by a single observer who was blinded to the intervention.