The liver tissues from Balb/c mice infected with C.sinensis were analysed by immunohistochemistry using mouse anti-CsGRN sera (Fig. 4). Liver sections from infected and uninfected mice probed with naive serum were not shown here. Positive staining was indicated with brown. Compared with sections from normal mice, strong staining was detected in the bile duct and the liver tissue after 1 month of parasite infection. Additionally, the level of CsGRN was higher with extended time. Most importantly, brown granules were found around the cell nuclei, explaining that CsGRN entered the biliary epithelium cells and hepatic cells across the cell membrane (Fig. 4). CsGRN was also seen in the bile ducts that were distant from the liver flukes, while normal mouse biliary epithelium and liver tissues were unstained (Fig. 4a, c, e, g, i, k).Fig. 4Immunohistochemical localisation of CsGRN in infected Balb/c mouse livers. Sections of Balb/c mouse livers infected with C. sinensis treated with mouse anti-CsGRN serum (b, d, f, h, j and l) or mouse naïve serum (a, c, e, g, i and k). During early infection (1 month), CsGRN was observed mainly surrounding the bile ducts where parasites reside, and brown granules were found around cell nuclei, which explained how CsGRN entered the biliary epithelium cells across the cell membrane (d). From 2 months post-infection, CsGRN was also seen within the hepatocytes (f, h, j and l). With prolonged infection, more CsGRN gathered in bile duct epithelial cells and hepatocytes. Positive hepatocytes and biliary epithelium cells were even found in the smaller bile duct where adult worm could not reach. a, b normal, c, d 1 month, e, f 2 months, g, h 3 months, i, j 4 months and (k, l) 6 months post-infection. Arrows indicate positive cells. The images were magnified at 1,000× (scale-bar: 20 μm)