Masson trichrome staining

The lungs of mice were fixed with 4% paraformaldehyde and embedded in paraffin. For histological analysis and detection of collagen deposition, successive 5-µm lung sections were placed on slides and subjected to staining with trichrome stain (Masson) kits (Sigma-Aldrich, Germany), according to the manufacturer's instructions. Collagen content was determined by the ratio of collagen surface area (blue) to total surface area (red). Image J software was used to evaluate collagen deposition.