Fig. 3

Electron microscopy (a-b) and immunohistology (c-d) of heart tissue from recipient 2. a-b Transmission electron microscopy representative examples demonstrating particles whose morphology is compatible with coronavirus particles (arrows). c-d Absence of immunoreactivity for the viral nucleocapsid protein (c) which contrasts with the intense staining in the positive control (lung tissue specimen of a SARS-CoV-2 infected hamster) (d). Transmission electron microscopy. Tissues were fixed at 4 °C in 4% glutaraldehyde (Laborimpex, Brussels, Belgium) in phosphate buffer at pH 7.4 and postfixed in 1% osmium tetroxide (Laborimpex, Brussels, Belgium) for 1 H at 4 °C. They were then dehydrated in graded (70, 90, 100%) ethanol solutions (VWR International, Leuven, Belgium) and propylene oxide (Laborimpex, Brussels, Belgium), embedded in epon (SERVA, Zandhoven, Belgium) and hardened at 60 °C. Semi-thin sections were stained with 0.5% toluidine blue and examined by light microscopy. Ultra-thin sections (80 nm) were stained with uranyl acetate (Fluka, Bornem, Belgium) and lead citrate (Leica, Aartselaar, Belgium). These sections were examined using an EM 910 transmission electron microscope (60 kV) (Zeiss, Belgium). Immunohistology Sections were deparaffinized and rehydrated in graded alcohols. After endogenous peroxydase inhibition with H2O2 (3%), nonspecific binding sites were blocked with the Protein Block Serum-Free solution (Dako). Slides were incubated for 1H at room temperature with a rabbit polyclonal antibody against the SARS-CoV-2 recombinant fusion nucleoprotein (ABclonal). Immunodetection was performed with the Polyview plus AP-Rabbit and alkaline phosphatase (Enzo Life Sciences)