Bond rxm platform

For mouse Ace2 mRNA RNAscope ISH, an antisense probe targeting mouse Ace2 (GenBank accession number NM_001130513.1; catalog number 863818) with no cross-reactivity to hACE2 was used. For mouse Tmprss2 mRNA RNAscope ISH, an anti-sense probe targeting mouse Tmprss2 (GenBank accession number NM_015775.2; catalog number 496728) was used. The RNAscope ISH assay was performed using the RNAscope 2.5 LSx reagent kit (Advanced Cell Diagnostics, Newark, CA) on the automated BOND RXm platform (Leica Biosystems, Buffalo Grove, IL) as described previously (35 (link)).

For immunohistochemistry (IHC), 4-μm sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost Plus slides and subjected to IHC using an anti-nucleocapsid rabbit monoclonal antibody (HL344, Cell Signaling Technology, Danvers, MA). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems, Wetzlar, Germany). Following automated deparaffinization, heat-induced epitope retrieval was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody (diluted at 1:1,600 in primary antibody diluent [Leica Biosystems]) for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 min), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60°C oven for 30 min and mounted with a permanent mounting medium (Micromount, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.

For IHC, four-micron sections of formalin-fixed paraffin-embedded tissue were mounted on positively charged Superfrost Plus slides and subjected to IHC using anti-nucleocapsid rabbit polyclonal antibody (3A, developed by our laboratory) with the method previously described (80 (link)). IHC was performed using the automated BOND-RXm platform and the Polymer Refine Red Detection kit (Leica Biosystems). Following automated deparaffinization, heat-induced epitope retrieval (HIER) was performed using a ready-to-use citrate-based solution (pH 6.0; Leica Biosystems) at 100°C for 20 min. Sections were then incubated with the primary antibody [anti-SARS-CoV-2 nucleocapsid rabbit polyclonal antibody diluted at 1:5,000 in primary antibody diluent (Leica Biosystems)] for 30 min at room temperature, followed by a polymer-labeled goat anti-rabbit IgG coupled with alkaline phosphatase (30 min). Fast Red was used as the chromogen (15 min), and counterstaining was performed with hematoxylin for 5 min. Slides were dried in a 60°C oven for 30 min and mounted with a permanent mounting medium (Micromount, Leica Biosystems). Lung sections from a SARS-CoV-2-infected hamster were used as positive assay controls.