Sircol collagen assay.

The left lung was used for collagen quantification per manufacturer’s protocol (Bicolor, Life Science Assays). Briefly, left lung homogenate was shaken overnight at 4°C in 5 mL of 0.5 M acetic acid with 0.6% v/v pepsin. A total of 1 mL of dye reagent was added to 100 μL of transparent supernatant, and the samples were vortexed for 30 minutes. The residual pellet was washed by acid-salt wash buffer to eliminate unbound collagen, and pH was normalized with alkalization buffer. Absorbance was measured at a wavelength of 550 nm in a microplate reader. Measured collagen content was compared with a standard curve and represented as mg/mL of left lung homogenate.