Masson’s trichrome staining and fibrosis score.

The lung sections were stained with Ponceau (Sigma-Aldrich) and acid fuchsin for 5–10 min, treated with 1% phosphomolybdic acid for 5 min, and then counterstained with aniline blue solution (Sigma-Aldrich). All sections were subsequently dehydrated with ethanol, deparaffinized in xylene, and finally mounted with neutral balsam. For semiquantitative analysis of fibrosis by Ashcroft Score, four different levels separated by 100 μm were analyzed by two blinded independent investigators. Morphological changes in fibrotic lungs were quantified according to the criteria of Ashcroft et al. (4).