2.7 Pathological examination and virus protein antigen detection
Respiratory tract tissues of mice and lung tissues of humans were dehydrated, embedded, and serially sectioned with a thickness of 4 μm. The sections were stained with HE, and the pathological changes were observed under an optical microscope. An immunohistochemical method for the detection of viral protein was conducted as previously described (J. Wang et al., 2016). After antigen repairing, the primary nucleoprotein (NP) antibody (1:500) (kindly provided by the National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University) was added in sections. The sections were incubated overnight at 4°C, followed by the addition of goat anti-mouse IgG-specific biotin conjugate (Calbiochem) (1:50), development by DAB stain, and counterstaining slightly by hematoxylin. Human lung tissue sections infected with influenza virus H5N1 were used as a positive control, and 10% normal mouse serum was used as a MOCK control. The positive results were judged by the brownish coloring of the nucleus, and the cytoplasm around the nucleus appeared slightly brownish in color.