Sodium cacodylate

For transmission electron microscopy (TEM) analysis, 1 × 10⁸ sperm cells suspended in Tyrode’s albumin lactate pyruvate media [22 (link)] were inoculated with ASFV “ArmeniaΔ285L-GFPhuCD4” [14 (link)], gt II (MOI 5). Sperm cells were incubated with the virus suspension (48 h, 37°C, 5% CO₂, humidified atmosphere). A negative sperm cell culture control was treated similarly to verify appropriate handling and culture conditions.
Subsequently, inoculated sperm cells were washed three times in 1× PBS by centrifugation at 134× g, 10 min, 23 °C. The resulting pellet was treated with fixing solution (2.5% glutaraldehyde buffered in 0.1 M sodium cacodylate (pH 7.2), 300 mosmol, Serva Electrophoresis, Heidelberg, Germany) for at least 2 h at 4 °C, and embedded in 1.8% low-melting agarose (Biozym). Small pieces were postfixed in 1.0% aqueous OsO4, and stained en bloc with uranyl acetate. After stepwise dehydration in ethanol, cells were cleared in propylene oxide, infiltrated with Glycid Ether 100 (Serva Electrophoresis), and polymerized at 60 °C for 3 days. 60–70 nm ultrathin sections were prepared with an ultramicrotome (UC7, Leica Microsystems, Wetzlar, Germany) and collected on EM grids (300 mesh, Plano). Finally, the sections were counterstained with uranyl acetate and lead citrate and analyzed with a Tecnai-Spirit TEM (FEI) at an accelerating voltage of 80 kV.

The excised tumors were cut into small pieces (diameter: 1 mm). Next, the tumor tissue samples were fixed with 4% (v/v) formaldehyde (Carl Roth, Karlsruhe, Germany) and 2.5% (v/v) glutaraldehyde solved in sodium cacodylate (0.1 M and pH 7.2, Serva, Heidelberg, Germany) for 3 h at room temperature and then placed at 4 °C for overnight incubation. Next, tissues were washed with sodium cacodylate buffer (3 times for 10 min each) and further fixed for 2 h at 20 °C with 1% (w/v) osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA, USA). Next, an ascending ethanol series was employed to dehydrate tissue sections, and this was followed by staining the tissues with 2% (w/v) uranyl acetate diluted in 50% (v/v) ethanol. According to the manufacturer’s instruction, tissues were embedded in Araldite^® resin (Plano, Wetzlar, Germany). Afterwards, very thin sections were cut (70 nm in thickness) and mounted on carbon-coated 100 mesh grids (Qantifoil Mico Tools, Grossloebichau, Germany). The tissue sections were stained for 10 min with lead citrate and investigated at 80 kV in an electron microscope, Zeiss EM 902A (Carl Zeiss AG, Oberkochen, Germany).