B7151

Influenza virus titers were determined by the FFA in MDCK cells. Lung homogenates were serially diluted 10-fold with an infection medium and incubated with MDCK cells for 24 h at 37 °C with 5% CO₂. Thereafter, MDCK monolayers were washed twice with PBS and subsequently fixed with ice-cold 80% acetone for 15 min at room temperature. Viral foci were stained using a mouse monoclonal antibody against influenza A NP (nuclear protein), 1/2500 (MAB8258, Millipore, Burlington, MA, USA), secondary antibody anti-mouse IgG biotin conjugate, 1/4000 (B7151, Sigma-Aldrich, St. Louis, MO, USA), peroxidase labeled streptavidin, 1/4000 (Sigma-Aldrich S2438, USA) and 3-amino-9-ethylcarbazole (AEC; Sigma-Aldrich, St. Louis, MO, USA). Focus-forming units (FFU; NP-positive red-colored cells located apart from one another at a distance of two uncolored cells) were then calculated, and viral titers were expressed as FFU per mL.

Mouse ovaries used for histological analysis were fixed with 4% paraformaldehyde overnight at 4 °C, dehydrated, and embedded in paraffin. Ovarian sections (5-μm thickness) were incubated with anti-LC3 rabbit antibody (1:300; #L8918, Sigma-Aldrich), followed by incubation with a biotinylated secondary antibody (#B7151, Sigma-Aldrich) for 1 h at a dilution of 1:500. For lysotracker staining, ovarian sections pretreated as above were incubated for 2 min with 100 μM Lysotracker Red (Beyotime Institute of Biotechnology, Haimen, China) in phosphate-buffered saline (PBS). For H&E staining, the slides were stained with H&E after deparaffinization. The sections were dehydrated, mounted, and examined using a dotSlide digital virtual microscopy system (Olympus, Tokyo, Japan).