Chloramine t citrate buffer solution

A modified protocol was employed to measure hydroxyproline levels in tissue as previously described.^{17 (link),44 (link)} Briefly, weighed tissue samples were hydrolyzed and the supernatant was neutralized with 1% phenolphthalein and titrated against 10M NaOH. An aliquot was mixed with isopropanol and added to a chloramine-T/citrate buffer solution (pH 6.0) (Sigma). Ehrlich’s reagent solution was added and measured at 570 nm. Hydroxyproline levels were calculated by using 4-hydroxy-l-proline (Calbiochem) as standard, and results were expressed as μg hydroxyproline per weight of tissue that contained 10⁴ eggs.

Tissue samples were fixed in neutral buffered formalin, processed, and 5–7 μm sections stained with hematoxylin and eosin (H & E). Granuloma diameter of 20–50 granulomas per animal was determined using an ocular micrometer (Nikon NIS-Elements, Nikon Corporation, Tokyo, Japan). For fibrosis assessment, tissue sections were stained with chromotrope 2R and analine blue solution (CAB) and counterstained with Wegert's hematoxylin for collagen staining. Complementarily, a modified protocol of tissue hydroxyproline quantification was used [59 (link)]. In brief, weighed liver samples were hydrolyzed and the supernatant was neutralized with 1% phenolphthalein and titrated against 10 M NaOH. An aliquot was mixed with isopropanol and added to a chloramine-T/citrate buffer solution (pH 6.0) (Sigma). Ehrlich's reagent solution was added and measured at 570 nm. Hydroxyproline levels were calculated by using 4-hydroxy-L-proline (Calbiochem) as standard, and results were expressed as μg hydroxyproline per weight of liver tissue that contained 10⁴ eggs.

Hydroxyproline content as a measure of collagen production was quantified using a modified protocol [53 (link)]. In brief, weighed liver samples were hydrolyzed overnight at 110°C in 6 M HCl and then filtered through Whatman filter papers. Filtrate was neutralized with 1% phenolphthalein and titrated against 10 M NaOH. An aliquot was mixed with isopropanol and added to a chloramine-T/citrate buffer solution (pH 6.0) (Sigma). Ehrlich’s reagent solution (25 g p-dimethyl-amino-benzaldehyde, 37.5 ml 60% perchloric acid) was added and measured at 570 nm using a VersaMax microplate spectrophotometer (Molecular Devices). Hydroxyproline levels were calculated by using 4-hydroxy-L-proline (Calbiochem, San Diego, CA, US) as standard, and results were expressed as μg hydroxyproline per weight of liver tissue that contained 10⁴ eggs.