Envision flex target retrieval solution high

Immunohistochemistry (IHC) (Figs. 5C and  7D) was performed as previously described using an Autostainer Link 48 (Dako). The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 minutes at 97 °C for activation, and a mouse anti-SARS-CoV-2 N monoclonal antibody (clone 1035111, R&D Systems, Cat# MAB10474-SP, 1:400) was used as a primary antibody. The sections were sensitized using EnVision FLEX for 15 minutes and visualized by peroxidase-based enzymatic reaction with 3,3′-diaminobenzidine tetrahydrochloride (Dako, Cat# DM827) as substrate for 5 minutes. The N-protein positivity was evaluated by certificated pathologists as previously described. Images were incorporated as virtual slides by NDP.scan software v3.2.4 (Hamamatsu Photonics). The area of N-protein positivity was measured using Fiji software v2.2.0 (ImageJ).

Immunohistochemical (IHC) analysis (Fig. 6c and Supplementary Fig. 4) was performed as previously described^{2 (link),17 (link),26 (link),31 (link),35} using an Autostainer Link 48 (Dako). The deparaffinized sections were exposed to EnVision FLEX target retrieval solution high pH (Agilent, Cat# K8004) for 20 min at 97°C for activation, and a mouse anti-SARS-CoV-2 N monoclonal antibody (clone 1035111, R&D Systems, Cat# MAB10474-SP, 1:400) was used as a primary antibody. The sections were sensitized using EnVision FLEX for 15 min and visualized by peroxidase-based enzymatic reaction with 3,3’-diaminobenzidine tetrahydrochloride (Dako, Cat# DM827) as substrate for 5 min. The N protein positivity was evaluated by certificated pathologists as previously described^{2 (link),17 (link),26 (link),31 (link),35} . Images were incorporated as virtual slides by NDP.scan software v3.2.4 (Hamamatsu Photonics). The N-protein positivity was measured as the area using Fiji software v2.2.0 (ImageJ).

Glass slides containing paraffin-embedded samples of liver, lung and heart were heated at 60 °C, deparaffinized in xylene and rehydrated in ethanol. Antigen retrieval was performed by heating the tissue in a pressure cooker in the presence of EnVision Flex target retrieval solution, high pH (Agilent, Santa Clara, CA, USA). Endogenous peroxidase was blocked with hydrogen peroxidase in methanol (1:1). To reduce non-specific binding, samples were incubated for 10 min at room temperature using a protein blocker solution (Spring Bioscience, Pleasanton, CA, USA). The samples were then incubated overnight at 4 °C with anti-NS3 antibody produced in rabbit (1:200), produced in-house and provided by the Laboratory of Biotechnology and Structural Bioengineering of the Rio de Janeiro Federal University. Afterwards, the samples were incubated with anti-rabbit antibody horseradish peroxidase conjugate (Spring Bioscience, Pleasanton, CA, USA). Negative control samples were incubated only with the secondary horseradish peroxidase-conjugated antibody. Finally, the reaction was revealed with diaminobenzidine (Agilent, Santa Clara, CA, USA) as chromogen and sections were counterstained with Harris hematoxylin (Agilent, Santa Clara, CA, USA).