Vectamount permanent mounting medium h 5000

Paraffin sections were heated for 30 min at 60°C and treated with xylenes, followed by rehydration in decreasing concentrations of ethanol (100, 90, 80, and 70%). Antigen retrieval was performed by boiling in sodium citrate (pH 6.0) for 15 min, and peroxidase activity was blocked with hydrogen peroxide for 10 min. Blocking was performed in 2.5% normal horse serum (Vector Laboratories) and incubated in primary antibody overnight at 4°C. Mouse anti–SARS-CoV-2 spike antibody (clone 1A9; GeneTex GTX632604) was used at a dilution of 1:400. Secondary antibody and detection reagents from the VECTASTAIN Elite ABC-HRP Kit (PK-7200; Vector Laboratories) were used according to manufacturer instructions. Sections were counterstained with Hematoxylin QS (H-3404; Vector Laboratories), dehydrated in increasing concentrations of ethanol, cleared with xylenes, and mounted with VectaMount permanent mounting medium (H-5000; Vector Laboratories).

All chemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) except VECTASTAIN Elite ABC-Peroxidase kits (PK-7100) and VectaMount Permanent Mounting Medium (H-5000) for immunohistochemical staining, which were from Vector Laboratories (Burlingame, CA, USA). Primary antibodies included a rabbit anti-endothelial NOS (eNOS) antibody from BD Transduction Laboratories (San Jose, CA, USA; 610297) and a mouse anti-β-actin antibody from Santa Cruz Biotechnology (Dallas, TX, USA; sc-47778). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology; sc-2004) and goat anti-mouse IgG (Santa Cruz Biotechnology; sc-2005) antibodies for Western blotting, and biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) for immunohistochemistry.