Immunohistochemical Staining of Influenza Antigens

Rehydrated paraffin-embedded lung sections were treated with Antigen Unmasking Solution according the manufacturer’s instruction (Vector Laboratories Inc. Burlingame, CA, USA) to unmask the antigen. After blocking with 1% bovine serum albumin, the sections were incubated with mouse anti-influenza NP, rabbit anti-mouse CD4, or rabbit anti-mouse CD8 primary antibody (all from Abcam) at 4 °C overnight^{39 (link)}, followed by biotin-conjugated secondary antibody (Calbiochem, Darmstadt, Germany) for 30 min at room temperature. Streptavidin peroxidase complex reagent (Vector Laboratories, Burlingame, CA) was then incubated at room temperature for 30 min followed by color development with 3, 3′-diaminobenzidine (DAB, Vector Laboratories). The slides were mounted and examined under microscope. Representative images were captured with Olympus BX53 semi-motorized fluorescence microscope.