Hemagglutinin ha

The lung lobes of the animal in each experimental group were collected on days 0, 2, 3, 5, and 7 postinfection (p.i.), and were perfused with 10% neutral-buffered formalin (NBF) at 4°C for 48 hours. Then the fixed tissues were immersed in 30% sucrose in 0.1M PBS at 4°C overnight. Finally the tissues were embedded in paraffin and sections with 4μm thickness were obtained and staining with hematoxylin and eosin (H&E) or with immunohistochemical (IHC) staining for H7N9 virus by using hemagglutinin (HA, Sino Biological Inc., BeiJing, China), RIP3(Cell Signaling Technology Inc., MA, USA), MLKL and E-Cadherin (ABclonal, WuHan, China) at 4°C overnight, followed by incubation with HRP-conjugated goat anti-rabbit antibody (1:1000 dilution, KPL) at room temperature for 60 min. Finally, 3, 3′-diaminobenzidine tetrahydrochloride (DAB) was used for signal development, and 20% hematoxylin was used for counterstaining.
To calculate the numbers of immunopositive signals on the antibodies stained sections, five microscopic fields in every section around the bronchial tube were photographed randomly, and then manually outline the area of positive signals per fields. The percentage of the area with positive staining coverage was calculated via the Aperio ImageScope software.

The influenza vaccine contained a 5 μg dose of Influenza A H1N1 (A/California/04/2009) hemagglutinin (HA) (Sino Biological) and varied amounts of TLR7/8a, HPMC‐C12, and NPs. Consistent between all gel groups unless specifically altered: 2 wt% HPMC‐C₁₂: 10 wt% NP, conjugated NPs have 10% TLR7/8a conjugated PEG–PLA and 90% unconjugated PEG–PLA, 20 μg TLR7/8a total dose. For the PNP hydrogels, the vaccine cargo was added at the appropriate concentration into the PBS component of the hydrogel before adding the polymer and NP solutions, as described above. For Alum (Invivogen) vaccines, the formulation was prepared according to the manufacturer's instructions with a 5 μg dose of HA.