A1555

Immunohistochemistry (IHC) staining was performed using 3.5 μm sections. IHC was used to detect protein expression using AKR1C3 (1:100, A13568, Abclonal), CAV1 (1:100, A1555, Abclonal), AURKA (1:200, bs2749R, bioss), and TRIB3 (1:200, bs7538R, bioss) antibodies. Sections were dewaxed in xylene and rehydrated with graded concentrations of ethanol. Antigen retrieval was performed with sodium citrate antigen for 1.5 min and 3% hydrogen peroxide methanol solution for 30 min to inactivate endogenous horseradish peroxidase. Sections were then incubated with rabbit anti-human AKR1C3, CAV1, TRIB3, and AURKA antibodies. Sections were visualized using the Envision Detection System (Dako) and hematoxylin was used to stain the nucleus.

PIE monolayers were first incubated with SADS-CoV at an MOI = 200 for 30 min at 4°C, then transferred to 37°C, fixed with immune electron microscopy fixative (0.1% glutaraldehyde and 3%PFA in 0.1 M PBS) for 2 h at room temperature, and finally collected. The collected cell precipitate was resuspended and washed twice with precooled 0.1 M PBS (pH 7.4). After the supernatant was removed, samples were processed with dehydration, resin penetration, embedding and polymerization steps. Samples were sliced into 90-nm ultrathin cryosections using a Leica UC7 ultramicrotome and collected onto 150-mesh nickel grids for immunogold labeling. The nickel grids were incubated with a 1:20 dilution of rabbit anti-caveolin-1 antibody (A1555, ABclonal) overnight at 4°C. The nickel grids were rinsed with PBS 6 x 3 min and then incubated with a 1:100 dilution of gold-conjugated goat anti-rabbit IgG (G7402, Sigma Aldrich) for 2 h at 28°C. The grids were washed and stained with 2% uranyl acetate. Finally, the sections were examined on a transmission electron microscope (H-7650; Hitachi).