Ultrastructural Analysis of Infected Lung Tissue

Lung tissue from lethally infected animals was collected at 6 dpi and prepared for transmission electron microscopy. For ultrastructural analysis in ultrathin sections small pieces (∼1 mm³) of tissues were fixed for at least 1 hour in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.3, to which 0.03% picric acid and 0.03% CaCl₂ were added. Then they were washed in 0.1 M cacodylate buffer and post-fixed in 1% OsO₄ in 0.1 M cacodylate buffer, pH 7.3, for 1 hour, washed with distilled water and stained en bloc with 2% aqueous uranyl acetate for 20 min at 60°C. The samples were dehydrated in ethanol, processed through propylene oxide and embedded in Poly/Bed 812 (Polysciences, Warrington, PA). Semi-thin sections 1 µm thick were cut and stained with toluidine blue. Ultrathin sections were cut on Leica EM UC7 ultramicrotome (Leica Microsystems, Buffalo Grove, IL), stained with lead citrate and examined in a Philips 201 transmission electron microscope at 60 kV.