Aperio scanscope system at

Rat liver tissue samples were immediately fixed in 10% neutral buffered formalin at 4℃ and routinely processed for paraffin embedding and cross-sectioned to obtain 4 μm-thick sections with circular layer and myenteric ganglia cut longitudinally. Before use, sections were deparaffini-zed, rehydrated and processed for routine haematoxylin/eosin and histochemical staining.
The slides were incubated with a 0.1% Direct Red 80 (Sigma-Aldrich, Missouri, USA) dissolved in aqueous saturated picric acid (Sigma-Aldrich, Missouri, USA) for 1 hour, washed in acidified water (0.5% acetic acid (glacial). Dehydrated and mounted with VECTOR Mounting (VECTOR, California, USA). Collagen and non-collagen components were red- and orange-stained, respectively.
Digital images were captured with an Aperio Scanscope System AT (Leica, Wetzlar, Germany) and the area of fibrosis was quantified in 20×-magnification fields five area per specimen. An RGB (Red, Green, Blue) threshold was used to identify the areas of fibrosis using Imagescope software (Leica, Wetzlar, Germany). Fibrosis was expressed as percentage (%) of total area.

For IHC, formalin-fixed rat liver tissue sections were deparaffinized and rehydrated, then heated at 100℃ for 30 min in a HIER Citrate Buffer (0.01 M), (Zytomed, Berlin, Germany) for antigen retrieval. The sliced tissues were later dealt with Hydrogen Peroxide Block (Thermo, Massachusetts, USA) to block the endogenous peroxidase activity. Next, the slides were blocked with Ultra V Block (Thermo, Massachusetts, USA). After rinsing in wash buffer, the sections were incubated with anti-alpha 1 antitrypsin antibody (Abcam, Cat. No. ab9373, Cambridge, UK), rabbit polyclonal to human serum albumin (Abcam, Cat. No. ab2406, Cambridge, UK), and monoclonal mouse anti-human cytokeratin 18 (Dako, Cat. No. M7010, Santa Clara, USA) for 1 hour at room temperature. The slides were then rinsed in wash buffer and incubated for 15 min with secondary antibody horseradish peroxidase-conjuga-ted (HRP Polymer, Thermo, Massachusetts, USA). Immune complexes were detected through the standard substrate detection of HRP. Last, the slides were stained with hematoxylin and dehydrated in graded alcohols and xylene. Digital images were captured with an Aperio Scanscope System AT (Leica, Wetzlar, Germany).