Immunohistochemistry of Influenza-Infected Lung Tissue

Immunohistochemistry (IHC) was performed as described previously [48 (link), 50 (link)]. Briefly, the immunized mice (C57BL/6, female) were i.n. challenged with 10 LD₅₀ of PR8, 20 LD₅₀ of CA04, or 10 LD₅₀ of H3N2. Seven days after virus infection, the mice were sacrificed and lung tissues from the mice were obtained, fixed with 10% neutral-buffered formalin, and embedded in paraffin. Formalin-fixed, paraffin-embedded tissue was cut into 4 μm sections and mounted onto glass slides. Tissue slides were blocked with 10% normal goat serum in PBS for 1 h at RT, washed with PBS containing 0.1% Triton X-100 (PBSX), and stained with anti-influenza A NP antibody (Southern Biotech, USA) for 2 h at RT. After washing with PBSX, slides were stained with Alexa Fluor 488-conjugated goat anti-mouse IgG (Invitrogen) for 2 h at RT, followed by staining with 10 μg/ml of 4´,6-diamidino-2-phenylindole (DAPI; Sigma, USA) for 10 min at RT. Then, the stained tissue slides were examined using an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss, Germany).