Picrosirius red staining

Collagen deposition was determined by picrosirius red staining of paraffin-embedded lung sections or cultured cells. Briefly, 4 μm dried tissue sections or monolayers of 2%-paraformaldehyde (PFA)-fixed A549 and MRC-5 cells were stained with 0.1% direct red stain (365548, Sigma Aldrich)/0.5% picric acid (36011, Sigma Aldrich) for 1 h at room temperature (picrosirius red staining) [21]. Each slide was examined and analyzed remotely using Image J software (W.S. Rasband, National Institutes of Health, Bethesda, MD; http://rsb.info.nih.gov/ij/, 1997–2011), and the mean staining intensity was quantified with the threshold detection method on grayscale images taken from three different microscope fields (magnification: x20).

For cell monolayers, bound picrosirius red was eluted by incubation for 1 h at 37°C with 0.1 N NaOH. Solubilized dye was recovered after each treatment and the absorbance was measured at 550 nm. The results were normalized against the cell density in the remaining monolayers after staining with the DNA-binding dye Crystal Violet for 30 min at room temperature. After elution with pure methanol, the dye’s absorbance at 540 nm was measured [22].