Methylene blue tetrahydrate

The infectivity of the challenge influenza viruses was adjusted to 200 PFU/mL, and 100 μL of each virus was mixed with an equal volume of MLE. Mixtures were incubated at 35 °C for 30 min and spread on 30-mm culture plates containing monolayers of MDCK cells. Subsequently, cells were overlaid with 3 mL of 1% agar containing 15 μg/mL acetylated trypsin from bovine pancreas (Sigma-Aldrich, Germany). After 3 days at 35 °C in a 5% CO₂ incubator, the agar of the overlaid media was removed, and cells were fixed at room temperature for 1 h with 1 mL of 3.7% formaldehyde in phosphate-buffered saline (PBS), rinsed with water, and stained with 1 mL of methylene blue tetrahydrate (Wako) in distilled water. Finally, plates were rinsed with water and dried at room temperature, and plaques were counted.

Male Institute of Cancer Research (ICR) mice were purchased from Sankyo Labo Service Corporation, Inc. (Tokyo, Japan). Animals were housed in a temperature-controlled (24 ± 1 °C) facility maintained on a 12-h light:12-h dark cycle with standard food available ad libitum for 1 week. All animal care and use procedures were approved by the Tokyo University of Science Ethics Committee (approval numbers: Y14052, Y15042, Y16044).
Isoflurane used for anesthetizing the mice, PPE, ATRA, dimethyl sulfoxide (DMSO), Tween80, methylene blue tetrahydrate, Mayer’s hematoxylin solution, 0.5% eosin Y, ethanol solution, absolute ethanol, and saline were purchased from Wako Pure Chemical Industries (Osaka, Japan).