Scaleview a2 solution

Mice were killed and intracardially perfused with PBS to remove blood cells from the lung. The lungs were isolated after intratracheal perfusion with 4% PFA phosphate buffer solution. The lung tissues were cleared with SCALEVIEW-A2 solution (Olympus, Tokyo, Japan) according to the manufacturer’s instructions. Images were acquired by using a stereo fluorescence microscope (M205FA, Leica Microsystems, Wetzlar, Germany) equipped with a digital camera (DFC365FX, Leica Microsystems).

A total of 10⁵ PFU of MA-eGFP-PR8 was intranasally inoculated into B6 mice. To label lung macrophages, 50 μl of PE-CD11b (M1/70, BioLegend, San Diego, CA) was injected intravenously to the mice at day 3 p.i. Thirty minutes after the antibody injection, the lungs of the mice were harvested. The kinetics of eGFP- and PE-positive cells in the lungs was imaged with a two-photon laser microscope (LSM 710 NLO, Carl Zeiss, Oberkochen, Germany). During the analysis, the lungs were maintained in complete medium (RPMI 1640 with 10% fetal calf serum) in a humid chamber (37 °C, 5% CO₂). The data were processed with LSM software Zen 2009 (Carl Zeiss). For three-dimensional imaging of HPAI virus-infected lung tissues, B6 mice were intranasally inoculated with 10⁵ PFU of MA-Venus-HPAI virus and MA-Venus-PR8. The lung tissues were collected from the mice at day 1 and 2 p.i., and treated with SCALEVIEW-A2 solution (Olympus) to make tissues transparent as described above. Three-dimensional images of lung tissues were obtained from a two-photon laser microscope (LSM 710 NLO).