Abstract
Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.
Highlights
Collagen is one of the major component in the adult gastro-intestinal wall, starting from the early stages of organogenesis [1] up to normal adult life and pathological conditions, such as inflammatory-induced bowel fibrosis [2,3,4]
Staining protocols, carried out by van Gieson and Sirius Red, alone or combined with Fast Green, were compared for their capability of revealing and allowing the quantification of collagen deposition in paraffin sections of colon from normal rats or with colitis induced by 2,4-dinitrobenzenesulfonic acid (DNBS)
Great amounts of tightly packed collagen fibers were highlighted by Sirius Red staining at the level of tunica mucosa and submucosa (Fig 1E and 1F) as well as in the circular and longitudinal layers of tunica muscularis and along the myenteric ridge encasing most of the ganglia (Fig 1G and 1H)
Summary
Collagen is one of the major component in the adult gastro-intestinal wall, starting from the early stages of organogenesis [1] up to normal adult life and pathological conditions, such as inflammatory-induced bowel fibrosis [2,3,4]. Histochemistry represents a and quick procedure for detecting total collagen tissue content In this regard, picro-sirius red dyes are widely used due to their specific reactivity to most collagen types [5,6,7] and, they have been largely employed for quantitative estimations of tissue fibrosis [6,7,8] in several organs, such as liver [9,10,11], lung [12,13], kidney [14,15,16] and gastrointestinal tract [17,18]. Staining protocols, carried out by van Gieson and Sirius Red, alone or combined with Fast Green, were compared for their capability of revealing and allowing the quantification of collagen deposition in paraffin sections of colon from normal rats or with colitis induced by 2,4-dinitrobenzenesulfonic acid (DNBS)
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