Histopathological Evaluation of Influenza Infection

Mouse lungs were fixed in 4% paraformaldehyde and stained with hematoxylin and eosin (HE). Elastica van Gieson method [32] (link) was performed to visualize easily crumpling alveoli without air space because elastic fiber in alveolar septa was stained black. Masson's trichrome staining [33] (link) was performed to visualize hyaline membrane formation. Immunohistochemical analysis was performed to detect influenza virus antigens. In brief, the histopathological slides were deparaffinized and treated with methanol containing 3% hydrogen peroxide to block endogenous peroxidase. The slides were incubated with rabbit anti-human influenza A, B virus polyclonal antibody (code #M149) [34] (link) and were treated with HistoMouse Max (Invitrogen, San Diego, CA) in accordance with the manufacturer's instructions [35] (link). Histopathological evaluation was measured by microscope investigation. The slides were viewed using an Olympus BX51 microscope and software DP controller to capture images.