Hydroxyproline Quantification in Mice Lungs

Mice lungs were evaluated for hydroxyproline content as previously described [24 (link)]. Briefly, the sample was homogenized in 1 mL of PBS and hydrolyzed using 1 mL of HCl. The sample was centrifuged, and the supernatant was used for hydroxyproline assay. Citrate/acetate buffer (deionized distilled water supplemented with 238 mM Citric acid, Sigma-Aldrich; 1.2% glacial acetic acid, Sigma-Aldrich; 532 mM sodium acetate, Sigma-Aldrich; and 850 mM sodium hydroxide, Nacalai Tesque) and chloramine T solution (1.0 mL deionized distilled water supplemented with 0.141 g chloramine T, Sigma-Aldrich; 1.0 mL 1-propanol, Sigma-Aldrich; and 8.0 mL citrate/acetate buffer) were added. After incubation, Ehrlich’s reagent (2.5 g 4-dimethylaminobenzaldehyde, Sigma-Aldrich; 9.3 mL 1-propanol and 3.9 mL 70% perchloric acid; Sigma-Aldrich) was added. After incubation at 65°C for 30 minutes, the absorbance was measured using a plate reader (iMARK, Bio-Rad, Hercules, CA, USA).