Hydroxyproline assay

Lung tissue was hydrolyzed in 1 ml of 6 M HCl at 95°C overnight. The hydrolysate was cooled down to RT and centrifuged for 10 min at 13,000g. The black particles on the surface of the hydrolysate were removed by vacuum sucking. Two and a half microliters of each sample was added to an indicated well of assay plate. Hydroxyproline standard solution was purchased from Sigma-Aldrich. Standard solution (1 mg/ml) was diluted 10 times, and 0, 2, 4, 6, 8, and 10 μl of diluted standard solution were added into different wells in the assay plate for generation of standard curve. Then, the assay plate was placed in a 60°C oven to dry samples. One hundred microliters of freshly made chloramine-T solution (2.0 ml of n-propanol, 0.282 g of chloramine-T, and 2.0 ml of H₂O in 20 ml of citrate acetate buffer) was added into each well, and the mixture was incubated at RT for 20 min. Then, 100 μl of fresh Ehrlich solution (4.5 g of 4-dimethylaminobenzaldehyde in 18.6 ml of n-propanol and 7.8 ml of perchloric acid) was added into each well. After that, the assay plate was placed at a 60°C oven for 60 min before reading at 560-nm wavelength in a Thermax plate reader.