Ehrlich s solution

The collagen content of native and decellularized tissue was evaluated using hydroxyproline colorimetric determination. The freeze-dried tissue was transferred to a microtube and then immersed in 6 N HCl and hydrolyzed at 100 °C for 24 h. After hydrolysis, the cap was opened and the tubes were heated at 90 °C to remove the remaining HCl. Each sample was dissolved in 500 µL of assay buffer composed of 5 mg/mL citric acid (monohydrate), 12 mg/mL sodium acetate (trihydrate), 3.4 mg/mL sodium hydroxide, and 1.2 mL glacial acetic acid in distilled deionized water, and subsequently filtered through 0.45 µm PTFE (polytetrafluoroethylene). One-hundred µL of each sample was transferred to a 96-well plate and added to 50 µL of 62 mM chloramine-T solution in 53.3% assay buffer, 26% n-propanol, and 20.7% distilled deionized water. Samples were incubated at room temperature for 15 min and then mixed with 50 µL of Ehrlich’s solution (SIGMA-ALDRICH, Japan), and incubated at 60 °C for 30 min. The amount of hydroxyproline was measured at 550 nm using a microplate reader against a L-hydroxyproline standard curve (0–1000 µg/mL), which was then correlated with the collagen amount using a standard curve and a conversion factor of 7.46 mg collagen to 1 mg 4-hydroxyproline.

The hepatic collagen content was measured using the hydroxyproline assay as previously reported [22 (link)]. Briefly, 50 mg of liver tissue was homogenized in 250 μL of PBS. Then, 250 μL of the tissue homogenate was transferred to a glass vial containing 500 µL of 12 N HCl and incubated overnight at 120 °C in a dry bath incubator. The acid hydrolyzed homogenate was filtered using a 0.45 polyvinylidene fluoride (PVDF) filter. Then, 20 µL of sample and hydroxyproline standards were added to a 96-well plate and mixed thoroughly with 100 μL of Chloramine T solution at room temperature for 30 min. Finally, 100 μL of Ehrlich’s solution (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated at 65 °C for 15–20 min. The photometric product was determined using a microplate reader at wavelength 550 nm. The amount of hydroxyproline (µg) was calculated relative to the liver weight (g).

Animals were euthanized 21 days after injury with bleomycin (Fresenius; 3 U/kg), and lungs were perfused with 1X PBS and snap frozen in liquid nitrogen. These samples were then homogenized and incubated with 50% Trichloroacetic acid (Sigma, T6399) on ice for 20 mins followed by an overnight incubation at 110°C in 12N HCl (Fisher, A144). Samples were reconstituted in distilled water with constant shaking for 2h. Aliquots of these samples were allowed to react with 1.4% Chloramine T (Sigma, 85739) and 0.5 M sodium acetate (Sigma, 241245) in 10% 2-propanol (Fisher, A416). The samples were incubated with Ehrlich’s solution (Sigma, 03891) for 15 min at 65°C. hydroxyproline content was measured by the analyzing absorbance at 550 nm with respect to the standard curve generated from purchased hydroxyproline (Sigma, H5534).

An IDO enzymatic activity was performed using a protocol adapted from previously described methods [53 (link), 54 (link)]. Murine samples used in the IDO activity assays were prepared in Dulbecco's Phosphate Buffered Saline (DPBS) (Fisher) containing protease inhibitors followed by sonication. 2X IDO reaction buffer (200 mg/ml catalase from bovine liver (Sigma), 800 mM L-tryptophan (Sigma), 100 mM DPBS, 40 mM L-ascorbic acid (Sigma), 20 mM methylene blue (Fisher), with and without tryptophan, was added to samples that were normalized for total protein by BCA assay. Samples were incubated at 37°C for 30 min and the reaction was stopped with 30% trichloroacetic acid (TCA) (Sigma). Samples were incubated again at 52°C for 30 min followed by centrifugation (5 min, 10,000 rpm, 4°C). Supernatants were used for spectrophotometric analyses by the addition of an equal amount of Ehrlich's Solution (Sigma). Samples were read at an absorbance of 480 nm and values were calculated based on a standard curve of L-Kynurenine (Sigma) from 0-30,000 mM. Final IDO activity values were determined by taking the difference between samples without tryptophan-containing IDO reaction buffer and those receiving tryptophan-containing IDO reaction buffer.

Liver samples were homogenized in 6 N HCl, hydrolyzed at 100 °C for 18 h, and centrifuged at 9391xg for 5 min. The supernatant was dried in speed-vacuum, dissolved in H₂O, and neutralized with 10 N NaOH. Samples were incubated in a chloramine-T solution [60 mM chloramines-T (Sigma, 857319), 20 mM citrate, 50 mM acetate, pH 6.5] for 25 min at room temperatures, and then in Ehrlich’s solution (Sigma, 038910) at 65 °C for additional 20 min. Hydroxyproline content was measured using a Beckman Coulter AD 340 Plate Reader (570 nm) and normalized to liver weight.