Following euthanasia and cardiac perfusion with sterile saline, cerebellar and spinal tissue samples were immediately collected, fixed on 10% formalin for 7 days and processed for immunohistochemistry. Brightfield immunohistochemistry was performed following previously described procedures (Maximova et al., 2008). The following primary antibodies were used: WNV-specific primary antibodies in hyperimmune mouse ascitic fluid (ATCCVR-1267 AF; 1:1000); anti-synaptophysin (mouse monoclonal [SY38]; Abcam; 1:10); anti-MAP2 (mouse monoclonal [5F9]; Millipore-Sigma; 1:6000); anti-CD68 (mouse monoclonal [KP1]; Biocare Medical; 1:500); anti-GFAP (rabbit polyclonal; Agilent; 1:4000); anti-CD4 (mouse monoclonal; Biocare Medical; 1:10); anti-CD8 (rabbit polyclonal; Abcam; 1:400); and anti-CD20 (mouse monoclonal; Agilent; 1:200). Diaminobenzidine was used for colorimetric detection (brown) of each protein marker. Sections were counterstained with hematoxylin. Whole tissue section imaging was performed at ×40 magnification using the ScanScope AT2 (Leica Biosystems). Aperio eSlide Manager and ImageScope software were used for digital slide organization, viewing, acquisition, and analysis. Double immunofluorescent staining to identify WNV-infected neuronal cell types was performed using Bond RX (Leica Biosystems) according to manufacturer protocols and with the following primary antibodies: WNV-specific primary antibodies in hyperimmune mouse ascitic fluid (ATCCVR-1267 AF; 1:1000) (for WNV-antigens) and Calbindin 28K (rabbit polyclonal; Millipore-Sigma; 1:1000) (for Purkinje cells) or ChAT (rabbit monoclonal; Abcam [clone EPR16590]; 1:1000) (for spinal motor neurons) and host appropriate secondary antibodies labeled with red fluorescent dye Alexa Flour 594 (Life Technologies; 1:300) or biotinylated secondary antibody (Vector Laboratories; 1:200) and green fluorochrome streptavidin 488 (Life Technologies; 1:500) and DAPI nuclear counterstain (Vector Laboratories).

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