One part of the mouse liver was fixed in formalin for histological analyses and the remaining part was snap frozen in liquid nitrogen using Wollenberger tongs and stored at −80 °C for subsequent metabolomic analyses. The liver histological examinations were performed by an experienced liver pathologist (X.Z.) in a blinded manner according to the NASH Clinical Research Network scoring system (24 (link)). Liver fibrosis was quantified biochemically using a hydroxyproline colorimetric assay (Biovision).
Hydroxyproline colorimetric assay
Manufactured by Abcam
Sourced in United States
The Hydroxyproline Colorimetric Assay is a laboratory tool used to quantify the amount of hydroxyproline, an amino acid found in collagen. This assay provides a colorimetric detection method for measuring hydroxyproline levels in various sample types.
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4 protocols using hydroxyproline colorimetric assay
1
Histological and Metabolomic Analysis of Mouse Liver
2
Aging Exacerbates Pulmonary Fibrosis in Mice
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Specific pathogen-free (SPF) C57 BL/6N male mice (young mice, 2–4 months old; aged mice, 18–20 months old) were purchased from the HFK Bioscience company (Beijing, China). All animals were kept under the SPF environment at Tongji Medical College. All procedures for animal experiments were approved by the Animal Care and Use Committee of Tongji Hospital.
The animal model of pulmonary fibrosis was established by single intratracheal administration of BLM (2 mg/kg, MCE, USA) or normal saline (NS) as control. Mice were grouped as follows: NS (young mice intratracheal with NS), aged NS (aged mice intratracheal with NS), BLM (young mice intratracheal with BLM), and aged BLM (aged mice intratracheal with BLM). Mice were sacrificed 21 days after the establishment of the BIPF mice model. Bronchoalveolar lavage fluid (BALF) was carried out. Samples of lung tissue were collected. The left lung was paraffin embedded for hematoxylin-eosin (HE) staining and masson trichrome (MT) staining (21 (link)). Lung hydroxyproline contents were measured by hydroxyproline colorimetric assay (Biovision, Milpitas, USA).
The animal model of pulmonary fibrosis was established by single intratracheal administration of BLM (2 mg/kg, MCE, USA) or normal saline (NS) as control. Mice were grouped as follows: NS (young mice intratracheal with NS), aged NS (aged mice intratracheal with NS), BLM (young mice intratracheal with BLM), and aged BLM (aged mice intratracheal with BLM). Mice were sacrificed 21 days after the establishment of the BIPF mice model. Bronchoalveolar lavage fluid (BALF) was carried out. Samples of lung tissue were collected. The left lung was paraffin embedded for hematoxylin-eosin (HE) staining and masson trichrome (MT) staining (21 (link)). Lung hydroxyproline contents were measured by hydroxyproline colorimetric assay (Biovision, Milpitas, USA).
3
Liver Tissue Analysis Protocol
4
Hydroxyproline Quantification in WAT
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Hydroxyproline measurement was performed using a hydroxyproline colorimetric assay (BioVision, Waltham, MA, USA) [15 (link)]. Snap frozen WAT explants were weighted and homogenized in water (100 mL water/10 mg WAT explants) using tissue homogenization (Precellys 24 Tissue Homogenizer, Bertin technologies, Montigny-le-Bretonneux, France). Homogenates (100 µL) were heated with 100 µL 12 M HCl for 3 h at 120 °C; then, 10 µL of the supernatant and a hydroxyproline standard were evaporated before being incubated with chloramine-T and p-dimethyl amino-benzaldehyde (DMAB) reagent at 60 °C for 90 min. The absorbance was read at 560 nm and the concentration was calculated using the standard curve and weights from WAT explants.
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