Histological Evaluation of Lung Inflammation

Lung tissues were fixed with formalin, embedded in paraffin, sectioned, and stained with hematoxylin-eosin (HE). Inflammatory responses were evaluated by scoring the microscopic findings in 20 visual fields of 12 different sections for each group, using the following criteria regarding the bronchus or bronchial area: 2 for mucus occlusion in the bronchial space, 1 for swelling of alveolar–epithelial cells, infiltration of inflammatory cells in the peri-bronchus, destruction of bronchial epithelial cells, or bleeding in the bronchial space. The following scoring method for the lung parenchyma was employed: 3 for destruction of the alveolar structure of >50%, 2 for that of 25–50%, and 1 for that of <25%, thickening of the alveolar wall, infiltration of inflammatory cells, or bleeding in the alveolar space [32 (link)]. Microscopic images were taken using a Life Technologies EVOS XL Core light microscope at 40× magnification.
Expression of influenza HA antigen was confirmed by staining with monoclonal antibody against influenza H1N1 pandemic HA antigen (ab66189, abcam, Cambridge, UK), goat antibody against mouse IgG conjugated with HRP (ab6789, abcam,), and DAB substrates (Invitrogen, Carlsbad, CA, USA).