Ab66189

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).

Vero cells were infected with MVAIK/PdmHA, fixed with 1% glutaraldehyde, and stained with a 1:500 dilution of monoclonal antibody against measles N protein (Ceminon, Temecula, CA, USA) and then stained with goat anti-mouse IgG conjugated with Alex Flour 488 (ab150113, abcam, Cambridge, UK). B95a cells were infected with MVAIK/PdmHA or empty MVAIK vector virus and stained with monoclonal antibodies against measles HA protein and goat anti-mouse IgG conjugated with Alexa Flour 488 (ab150113, abcam, Cambridge, UK).
To investigate the expression of influenza HA protein in Vero and B95a cells, monoclonal antibody against influenza A/H1N1 HA protein (ab66189, abcam, Cambridge, UK) and secondary antibody against mouse IgG conjugated with Alexa Flour 568 (ab175473, abcam, Cambridge, UK) were used for influenza H1N1 HA staining.

Infected Vero cells were analyzed using a scanning electron-microscope (SEM), monoclonal antibody against influenza A/H1 HA protein (ab66189, abcam, Cambridge, UK), and secondary antibody against mouse IgG conjugated with 50 nm Au colloid (Cytodiagnostics, Toronto, Canada). Samples were fixed, dehydrated, dried, and coated with 2-nm-thick platinum–palladium using a JFC-1200 Fine Coater (JEOL, Tokyo, Japan) with an accelerating voltage of 13 kV.