A039829 2

Fluorescent multiplex immunohistochemistry (FMIHC) was performed with the Tyramide Signal Amplification (PerkinElmer) system using an Opal IHC kit (Akoya Biosciences, Inc.), according to the manufacturer's instructions, as previously described.²¹ The antibodies used in this procedure included MPO (A039829‐2; Dako) and anti‐histone H3 (citrulline R2 + R8 + R17) (ab5103; Abcam). We used a fluorescence microscope (Keyence; BZ‐9000) to capture images (Keyence BZ‐II Analyzer Ver 1.41). FMIHC was performed on representative slides containing DN or GN and the co‐expression of MPO and anti‐citH3 was observed in the cell detritus of necrotic foci.

Nasal turbinate and lung tissue samples were harvested from hamsters infected with SARS-CoV-2 at 2 or 4 dpi. Tissue samples were fixed in 10% phosphate-buffered formalin, and nasal turbinate was decalcified with 10% EDTA solution (pH 7.0). Tissue samples were then embedded in paraffin. The paraffin blocks were sectioned at 4-μm thickness and mounted on Platinum PRO micro glass slides (Matsunami, Osaka, Japan). For histopathological analysis, slides were stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, slides were heated in citrate buffer for 5 min using a pressure cooker for antigen retrieval and blocking with Block Ace (KAC, Kyoto, Japan), followed by staining with anti-SARS-CoV-2 spike antibody (GTX632604; GeneTex, Hsinchu, Taiwan), anti-SARS-CoV-2 nucleocapsid antibody (GTX635679- GeneTex), anti-CD3 (ab16669; Abcam, Cambridge, UK), antimyeloperoxidase (MPO) (A039829-2; Dako; Agilent, Santa Clara, CA), or anti-Iba1 (019-19741, Fujifilm, Wako, Osaka, Japan). Immunostaining was detected by EnVision system peroxidase-labeled anti-rabbit or anti-mouse immunoglobulin (Dako) and visualized with a Histofine diaminobenzidine substrate kit (Nichirei Biosciences, Tokyo, Japan).