Discovery cc1

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.

IHC was performed on 4 µm formalin-fixed, paraffin-embedded tissue sections using BRG1 (SMARCA4), AR, FOXA1, ERG and C-Myc. The IHC process was performed on the Ventana ULTRA automated slide staining system using the Omnimap and Ultraview Universal DAB detection kit. The antibody details are provided in SI Appendix, Table S1. The following commercial kits from Roche-Ventana Medical System were used: Discovery CC1 (Cat No. 950-500), Discovery CC2 (Cat No. 950-123), OptiView Universal DAB Detection Kit (Cat No. 760-700), and OmniMap Universal DAB Detection Kit (Cat No. 760-149).

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.

Immunohistochemistry (IHC) was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections using BRG1 (SMARCA4), AR, FOXA1, ERG and C-Myc. The IHC process was performed on the Ventana ULTRA automated slide staining system using the Omnimap and Ultraview Universal DAB detection kit. The antibody details are provided in the SI Appendix, Table S1. The following commercial kits from Roche-Ventana Medical System were used: Discovery CC1 (Cat No. 950-500), Discovery CC2 (Cat No. 950-123), OptiView Universal DAB Detection Kit (Cat No. 760-700), and OmniMap Univesral DAB Detection Kit (Cat No. 760-149).

Immunohistochemistry staining was performed using the Discovery ULTRA automated stainer from Roche Diagnostics (Indianapolis, IN). In brief, antigen retrieval was performed using a tris/borate/EDTA buffer (Discovery CC1, 06414575001; Roche), pH 8.0 to 8.5, at 95 °C for 32 min. For AR staining only, 64 min of antigen retrieval time was applied. The slides were then incubated with primary antibodies for 1 h at room temperature with the following dilutions: TMPRSS2 (ab92323), 1:3000; TMPRSS2 (ab214462), 1:200; Androgen Receptor (ab133273), 1:100; ACE2 (R&D Systems, #AF933), 1:400. The antibodies were visualized using the OmniMap anti-Rabbit HRP (05269679001; Roche), and OmniMap anti-Goat HRP (06607233001; Roche) in conjunction with the ChromoMap DAB detection kit (05266645001; Roche). Lastly, the slides were counterstained with hematoxylin and bluing. The specificity of each antibody was first tested on appropriate control tissues (Fig. S3) before proceeding to staining of the lung sections. Controls without primary antibody were included in all experiments (Fig. S4).