Hrp multimer cocktail

Fetal brains were harvested in 4% PFA and stored at 4 °C. The tissue was dehydrated, fixed and infiltrated with paraffin on the Leica Polaris 2 tissue processor. The infiltrated tissue was embedded and sectioned at 5 microns. Antigen retrieval was performed on the Roche Ventana Discovery ULTRA staining platform using Discovery CC1 (Roche cat#950-500) for a total application time of 64 min. The primary antibody, human anti-Zika-NS1(EB9) (2 mg/mL), was incubated as a 1:400 dilution, at room temperature, for 44 min. Secondary immunostaining used a horseradish peroxidase (HRP) multimer cocktail (Roche cat#760-500) and immune complexes were visualized using the ultraView Universal DAB (diaminobenzidine tetrahydrochloride) Detection Kit (Roche cat#760-500). Slides were then washed with a Tris based reaction buffer (Roche cat#950-300) and counter-stained with Hematoxylin II (Roche cat #790-2208) for 4 min.

Hamster lungs were harvested in 4% PFA and stored at 4 °C. The tissue was dehydrated, fixed, and infiltrated with paraffin on the Leica Polaris 2 tissue processor. The infiltrated tissue was then embedded and sectioned at 5 microns. Antigen retrieval was performed on the Roche Ventana Discovery ULTRA staining platform using Discovery CC1 (Roche, Indianapolis, IN, USA; Cat#950-500) for a total application time of 64 min. The primary antibody, human Anti-SARS-CoV-2 Nucleocapsid monoclonal (E16C) (Thermo Fisher, Corning, NY, USA; MA1-7403, 0.1 mg/mL), was incubated at a 1:100 dilution, at room temperature, for 45 min. Secondary immunostaining used a horseradish peroxidase (HRP) multimer cocktail (Roche, Indianapolis, IN, USA; Cat#760-500), and immune complexes were visualized using the ultraView Universal DAB (diaminobenzidine tetrahydrochloride) Detection Kit (Roche, Indianapolis, IN, USA; Cat#760-500). Slides were then washed with a Tris-based reaction buffer (Roche, Indianapolis, IN, USA; Cat#950-300) and counter-stained with Hematoxylin II (Roche, Indianapolis, IN, USA; Cat #790-2208) for 4 min. Scoring was performed as shown in Table S2.