Frozen (−80 °C) small intestinal tissues including duodenum, jejunum and ileum taken from the experimentally infected pigs were pre-frozen at −20 °C for 10 min. Tissues were then embedded in optimal cutting temperature (OCT) compound and cut into 8-µm sections using the Cryotome FSE machine (Thermo Fisher Scientific). Mounted microscope slides were fixed with paraformaldehyde and stained with haematoxylin and eosin for histopathological examination.
For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).
For immunohistochemistry analysis, a rabbit antibody raised against the SADSr-CoV 3755 N protein was used for specific staining of SADS-CoV antigen. Slides were blocked by incubating with 10% goat serum (Beyotime) at 37 °C for 30 min, followed by overnight incubation at 4 °C with the rabbit anti-3755 N protein serum (1:1,000) and mouse anti-cytokeratin 8+18+19 monoclonal antibody (Abcam), diluted 1:100 in PBST buffer containing 5% goat serum. After washing, slides were then incubated for 50 min at room temperature with Cy3-conjugated goat-anti-rabbit IgG (Proteintech) and FITC-conjugated goat-anti-mouse IgG (Proteintech), diluted 1:100 in PBST buffer containing 5% goat serum. Slides were stained with DAPI (Beyotime) and observed under a fluorescence microscope (Nikon).