Apoptosis of lung tissue was detected using the TUNEL assay with an Apoptosis Assay kit (Roche Diagnostics GmbH). The lung tissue slices were naturally dried, dewaxed with xylene and dehydrated with a gradient of alcohol solutions. The tissues were then added to 50 µl TdT enzyme reaction solution and incubated at 37°C for 60 min in the dark, then washed with PBS. Subsequently, tissue slices were added to 50 µl Streptavidin-TRITC labeling solution and incubated in a wet box at 37°C for 30 min. After washing with PBS, tissues were counterstained with DAPI solution, incubated for 15 min, sealed with mounting medium and observed under a fluorescence microscope (Olympus IX71; Olympus Corporation). TUNEL-positive cells in the images were counted using ImageJ software (version 6.0; National Institutes of Health).

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