Anti sense probes

SARS CoV2 RNA was visualized by RNAscope in situ hybridization with anti-sense probes and reagents from Advanced Cell Diagnostics as previously published (Deleage et al., 2016 (link)). Briefly, slides were boiled in RNAscope Pretreat citrate buffer for 15 minutes. Pretreat reagent 3 (protease solution) was added at a 1:15 dilution, and the slides were incubated for 20 minutes at 40°C in the hybridization oven. SARS-CoV-2 antisense probes VnCoV2019S 21631–23303 of NC 045512.2, which hybridizes specifically to the 5’ end of SARS-CoV-2-Spike RNA, and VnCoV-N 28275–29204 of MN908947.3, which cross-hybridizes to SAR-CoV and MERS N-RNA, were added for 2 hours before continuing with AMPs 1–6 from the RNAscope 2.5 Red detection kit. Warp red chromogen was added to visualize the RNA for 5 minutes. Some sections were then blocked with Peroxidizer 1 and Background Sniper before adding CD68 (diluted 1:300, Dako) overnight. Polink-2 Plus HRP Mouse (GBI Labs) was added according to the manufacturer’s directions. ImmPact DAB was added to visualize macrophages before counterstaining all slides with CAT Hematoxylin and bluing in TBST. A thin layer of Clear mount diluted 1:5 in DI water (Thermo Scientific) was added to the sections, allowed to dry, dipped in xylenes before mounting in Permount.

SARS-CoV-2 RNA was visualized by RNAscope in situ hybridization (ISH) with antisense probes and reagents from Advanced Cell Diagnostics as previously published [12 (link)]. SARS-CoV-2 antisense probes VnCoV2019S 21631-23303 of NC 045512.2, which hybridizes specifically to the 5′ end of SARS-CoV-2 spike RNA, and VnCoV-N 28275-29204 of MN908947.3 were added before continuing with the RNAscope 2.5 Red detection kit. Warp Red chromogen was added to visualize the RNA.