Rnascope zz probe technology

Paraffin-embedded tissues from days 3, 5, and 14 pi were sectioned at 5 μm and subjected to in situ hybridization (ISH) using the RNAscope^® ZZ probe technology (Advanced Cell Diagnostics, Newark, CA). Tissues from inoculated and controls cats including nasal turbinate, palate/tonsil, retropharyngeal lymph nodes, trachea, lung, and heart were subjected to ISH using the RNAscope^® 2.5 HD Reagents–RED kit (Advanced Cell Diagnostics) following the manufacturer’s instructions, and using a probe targeting SARS-CoV-2 RNA spike (V-nCoV2019-S probe ref # 848561). A probe targeting feline host protein peptidylprolyl isomerase B (PPIB) was used as a positive control (Advanced Cell Diagnostics cat # 455011). A probe targeting DapB gene from Bacillus subtilis strain SMY was used as a negative control (Advanced Cell Diagnostics cat # 310043). Tissue sections were ISH scored based on labeling extension using a scoring system as follow: score 1 = up to 2% of the tissue section positive; score 2 = from 2 to 5% positive; score 3 = from 5 to 15% positive; score 4 = from 15 to 25%; score 5 = more than 25%; and score 0 = no labeling. Vero E6 cell infected with 0.1 MOI of SARS-CoV-2 D614G (B.1 lineage), Delta (B.1.617.2 lineage), or the Omicron BA.1.1 (B.1.1.529) and fixed at 24 h pi were used as positive controls (Suppl. Fig 1).

Paraffin-embedded tissues were sectioned at 5 µm and subjected to ISH using the RNAscope ZZ probe technology (Advanced Cell Diagnostics, Newark, CA). In situ hybridization was performed to detect tissue distribution of SARS-CoV-2 RNA in tissues. Palatine tonsil, mRPLN, and lung were tested by RNAscope 2.5 HD Reagents–RED kit (Advanced Cell Diagnostics) as previously described^{27 (link)}. Proprietary ZZ probes targeting SARS-CoV-2 RNA (V-nCoV2019-S probe) designed and manufactured by Advance Cell Diagnostics were used for detection of viral RNA. As positive controls, a probe targeted to the Bos taurus–specific cyclophilin B (PPIB) gene and a non-species-specific probe targeting the ubiquitin (UBC) gene were used. As a negative control, a probe targeting dapB of Bacillus subtilis was used.