Fast red dye

Histopathologic examinations were conducted, as described previously [31 (link)]. Briefly, paraffin-embedded tissues were deparaffinized and hydrated with xylene and alcohols, followed by staining with hematoxylin and eosin and investigation of microscopic lesions in mouse lungs. For IHC analysis, lung tissues were incubated with an anti-IAV antibody (Merck, Kenilworth, NJ, USA) and a biotinylated secondary antibody (Dako; Agilent Technologies, Santa Clara, CA, USA) at 37°C. After attaching with streptavidin alkaline phosphatase (Calbiochem, San Diego, CA, USA), treated tissues were colored using fast red dye (Sigma-Aldrich, St. Louis, MO, USA).

The cells were fixed with 2% paraformaldehyde in PBS, pH 7.0, for 15 min and incubated in a solution containing 10 ml 0.02 M Tris-HCl buffer pH 8.7, 1 mg Naphtol-AS-B1-phospate and 5 mg Fast Red dye (all from Sigma-Aldrich) at 37°C for 1 h.