Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

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.

Fig 1. Representative photomicrographs of full-thickness normal rat colon showing the distribution pattern of collagen fibers stained with van Gieson (VG, A-D), Sirius Red (SR, E-H) and Sirius Red/Fast Green (SR/FG, I-L).

CM and LM: circular and longitudinal muscle, respectively; MP: myenteric plexus. Scale bars represent 50 μm. Scatter plots show the percentage of positive pixel (PPP) of collagen ± SEM obtained from 6 rats in the whole colonic wall (M) and tunica muscularis (N). *,° P = 0.028 versus VG and SR, respectively.

Fig 2. Representative photomicrographs of haematoxylin/eosin-stained full-thickness colonic samples from normal rats (A,B), or rats with DNBS-induced colitis at day 6 (C,D).

Normal colon displays a normal morphological architecture of the wall and myenteric ganglia. In the inflamed colon, the following alterations are evident: infiltrated tunica submucosa; thickened and infiltrated tunica muscularis and serosa; vacuolated myenteric ganglia with altered cells and abundant eosinophilic infiltrations along the myenteric ridge (D, arrows and arrowheads, respectively). Scale bars represent 50 μm.

Fig 3. Representative photomicrographs of full-thickness inflamed rat colon showing the distribution pattern of collagen fibers stained with van Gieson (VG, A-D), Sirius Red (SR, E-H) or Sirius Red/Fast Green (SR/FG, I-L).

CM and LM: circular and longitudinal muscle, respectively; MP: myenteric plexus. Scale bars represent 50μm. Scatter plots show the percentage of positive pixel (PPP) of collagen ± SEM obtained from 6 rats in the whole colonic wall (M) and tunica muscularis (N). *,° P = 0.028 versus VG and SR, respectively.