Pre-Treatment with Zirconia Nanoparticles Reduces Inflammation Induced by the Pathogenic H5N1 Influenza Virus

Huo, Caiyun; Xiao, Jin; Xiao, Kai; Zou, Shumei; Wang, Ming; Qi, Peng; Liu, Tianlong; Hu, Yanxin

On days 3 and 6 post-infection, lung tissue samples from three mice per group were collected and fixed with 4% neutral formalin at room temperature for 48 h. Histopathological and viral antigen analyses of lung tissues on day 6 post-infection were performed using hematoxylin and eosin (H&E) staining and IHC staining, as previously described. 37 Briefly, serial tissue sections were cut to 5-μm thickness after embedding in paraffin. Each slide was stained with H&E and examined using light microscopy (Olympus CX31). For IHC staining, sections were incubated in 10% normal goat serum in PBS for 30 min to block non-specific binding sites prior to reaction with the anti-influenza nucleoprotein mAb (AA5H, Abcam) at 1:400 dilution in PBS for 2 h. The slides were further incubated with goat anti-mouse IgG conjugated with avidin (Chemicon, USA) for 1 h, followed by incubation in biotinylated peroxidase (Victoria, BC, Canada) for an additional 1 h. Staining was visualized through the addition of 3.3-diaminobenzidin (Sigma-Aldrich, St. Louis, MO, USA) for 15 min and counterstaining with hematoxylin; the slides were then mounted with neutral balsam.