Ap 9003 500

Both lungs, all lobes of the liver and PDAC metastases therein were collected during necropsy and fixed in formalin (Fisher, #SF-98-4) overnight before embedding in paraffin. Slides were cut on a microtome (5–7 μm) and deparaffinised before immunohistochemistry (IHC). Antigen retrieval was performed using citrate buffer, pH 6.0 (Thermo, #AP-9003-500) in a steamer for 40 min before cooling and washing with PBS. Peroxidase blocking was achieved using 3% hydrogen peroxide, 5% normal horse serum (Invitrogen, #26050070) and 1% normal goat serum (Invitrogen, #16210064) for protein blocking, and primary antibodies were diluted 1:100 in protein block solution. Secondary antibodies were diluted 1:500 in protein block, and colorimetric reaction was accomplished using diaminobenzidine (DAB, Vector Laboratories #SK-4105). Counterstaining was accomplished using Gill’s haematoxylin #3 (Fisher, #NC9964763), and slides were dried and coverslipped using Permount (Fisher, #SP-15-100).

Lungs from SARS CoV-2 WA1/2020 and Omicron infected macaques were evaluated on day 2 following challenge by histopathology^{16 (link)}. At the time of fixation, lungs were suffused with 10% formalin to expand the alveoli. All tissues were fixed in 10% formalin and blocks sectioned at 5 μm. Slides were incubated for 30–60 min at 65°C then deparaffinized in xylene and rehydrated through a series of graded ethanol to distilled water. Sections were stained with hematoxylin and eosin. For SARS-N immunohistochemistry, heat-induced epitope retrieval was performed using a pressure cooker on steam setting for 25 min in citrate buffer (Thermo Fisher Scientific, AP-9003-500), followed by treatment with 3% hydrogen peroxide. Slides were then rinsed in distilled water and protein blocked (Biocare, BE965H) for 15 min followed by rinses in 1× PBS. Primary mouse anti-SARS-CoV-nucleoprotein antibody (Sinobiological; 40143-MM05) at 1:1000, was applied for 60 min, followed by mouse Mach-2 HRP-Polymer (Biocare) for 30 min and then counterstained with hematoxylin followed by bluing using 0.25% ammonia water. Staining was performed using a Biocare intelliPATH autostainer. Blinded evaluation and histopathologic scoring of eight representative lung lobes from cranial, middle and caudal, left and right lungs from each monkey was performed by a board-certified veterinary pathologist (AJM).