Histological and Immunohistochemical Analysis of SARS-CoV-2 in Lung Tissue

Histological and immunohistochemical assays were conducted as described^{51 (link),55 (link)}. Immediately after euthanasia, the left lung lobe was harvested and fixed by submersion in 10% phosphate-buffered formalin for overnight, followed by submersion in 70% ethanol for up to one week, which was enough to disinfect the viruses. Fixed tissue was manually embedded in paraffin, and sections were prepared at 4 µm thickness. Tissue sections were stained with hematoxylin and eosin (Richard Allan Scientific, San Diego, CA). For immunohistochemistry, antigen retrieval was performed with antigen unmasking solution (pH 6.0 citrate buffer, H-3300, Vector Laboratories, Newark, CA), followed by peroxide incubation. The tissue sections were first incubated with primary antibody, SARS-CoV-2 nucleocapsid protein rabbit mAb (26369, Cell Signaling, Danvers, MA) for overnight, and then with secondary antibody, HRP-conjugated anti-rabbit IgG antibody (MP7401, Vector Laboratories). The signal was then amplified using a DAB staining kit (NC9567138, Fisher Scientific, Waltham, MA) and it was counterstained with hematoxylin. Finally, tissues were tiled scanned using an Aperio AT2 slide scanner (Leica Biosystems, Deer Park, IL), and images were captured with an Aperio ImageScope (Leica Biosystems, Deer Park, IL).