Histofine diaminobenzidine substrate kit

The collected tissue samples were fixed in 10% phosphate-buffered formalin (pH 7.2) and embedded in paraffin. The sections were stained using Carazzi’s hematoxylin and eosin. For immunohistochemical analysis for the detection of WNV antigen, deparaffinized sections were subjected to antigen retrieval by 0.1% trypsin digestion at room temperature for 10 min. Then, the sections were incubated in 0.3% H₂O₂ in methanol (vol/vol) at room temperature for 15 min to quench endogenous peroxidase activity. After blocking with 10% normal goat serum for 10 min, the sections were incubated with primary antibodies at 4°C overnight, followed by 30 min incubation with biotinylated-secondary antibodies that were detected using the Histofine diaminobenzidine Substrate Kit (Nichirei, Tokyo, Japan).

Immunohistochemistry (IHC) was performed using the antibodies listed in Table 2. Following antigen retrieval, sections were treated with 10% normal serum for 15 min and then incubated with primary antibodies at 4 °C overnight. The signals were enhanced using the avidin-biotin complex method (Vector Lab, Burlingame, CA, USA). Color development was performed with 3, 3′-diaminobenzidine (Histofine diaminobenzidine substrate kit; Nichirei, Tokyo, Japan), and the staining results were observed with an optical microscope.
To investigate the relationship between the bone and fibrous stroma, we performed IHC staining of the samples from both groups. First, we stained collagen 1a, a marker of collagen fibers. Next, to check the bone-formation potential of the stroma, alkaline phosphatase (ALP) staining was performed.

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).