Figure 2. Immunohistochemical staining (peroxidase-antiperoxidase (PAP) technique) of liver samples from 2 rat-hepatitis E virus (HEV)–positive Norway rats from Germany, July 2009. Arrows indicate immunohistochemical positive reactions in the cytoplasm of single hepatocytes (A) and in a few foci in hepatocytes and stellate cells (B). For PAP staining, deparaffinized slides of liver samples were incubated with anti-HEV–positive human serum, which had been previously used to detect rat HEV by using solid phase immunoelectron microscopy (10), for 1 h at 37°C with protein A (Sigma-Aldrich, Steinheim, Germany) at a dilution of 1:100 for 45 min at 37°C and finally with PAP complexes from rabbits (Sigma, St. Louis, MO, USA) at a dilution 1:200 for 45 min at 37°C. AEC (3-amino-9-ethylcarbazol; Sigma Chemie GmbH, Deisenhofen, Germany) was used as the substrate chromogene. The slides were counterstained with hematoxylin and subsequently analyzed by light microscopy. Scale bars = 20 µm.