Fitc isolectin b4

Mouse tissue was cryopreserved using O.C.T. and cut into 5 µm sections. Cryosections were fixed in ice‐cold methanol/acetone. After blocking in 10% goat serum with 0.1% Triton X‐100, sections were incubated with primary antibodies diluted in 10% goat serum at 4°C overnight. Following primary antibodies were used: anti‐CD31 (550274, BD Biosciences, 1:50), anti‐cleaved caspase 3 (9664, Cell Signaling Technology, 1:100), anti‐ColIV (2150‐1470, Bio‐Rad, 1:250), anti‐ERG (ab93513, Abcam, 1:1,000), anti‐α‐SMA (C6198, Sigma‐Aldrich, 1:200), anti‐Ki‐67 (14‐5698‐82, Thermo Fisher Scientific, 1:100) or FITC‐Isolectin B4 (L2895, Sigma‐Aldrich, 1:100). DAPI and fluorophore‐conjugated secondary antibodies were added before embedding the sections in fluorescent mounting medium (DAKO). Images were acquired using a confocal microscope (Zeiss LSM 700) and analysis was done using Fiji software.

Pericytes and capillaries were stained in the granular layers of the cerebellar vermis and hemisphere slices as previously described [21 (link),42 (link)], focusing, respectively, on lobule V and lobule VI (see Supplementary Materials for details). The rabbit anti-NG2 chondroitin sulfate proteoglycan (Millipore, Merck KGaA, Darmstadt, Germany) and FITC-isolectin B4 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) primary antibodies were used to stain the pericytes and blood vessels, respectively. Slices were mounted on microscope slides, using ProLong^® Gold antifade reagent with DAPI (Molecular Probes, Thermo Fisher Scientifics, Waltham, MA, USA). Fluorescence of samples was observed with a TCS SP5 II LEICA confocal microscopy system (Leica Microsystems GmbH, Wetzlar, Germany) furnished with a LEICA DM IRBE inverted microscope. All acquisition files were visualized by LAS AF Lite software. Negative controls were carried out in parallel by treating slices with non-immune serum during the incubation procedures.

Mouse-4G2 (monoclonal antibody against anti-flavivirus group antigen, clone D1-4G2-4-15, ATCC), Hu-4G2 (recombinant monoclonal antibody of clone D1-4G2-4-15 with human IgG Fc produced in Nicotiana tabacum, a gift from Dr. Nobuyuki Matoba), anti-TTR (Abcam), anti-CD31 (Invitrogen), anti- PDGFβR (Abcam), anti-alpha smooth actin (Abcam), anti-Axl (R&D systems), FITC-isolectin B4 (Sigma Aldrich), Alexa Fluor 647-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch), Alexa Fluor 549-conjugated goat anti-human IgG (Jackson ImmunoResearch), and Alexa Fluor 488-conjugated goat anti-rat IgG (H+L) (ThermoFisher) were used for the immunofluorescence assay. All antibodies were validated for their specificity using mock or isotype antibody controls.
Mouse IFNAR-1 neutralizing antibody (clone MAR 5A3) and isotype control (clone MOPC-21) were purchased from BioXcell. anti-Axl (AF154), anti-GAS6 (AB885), and isotype control IgG (AB-108-C) were purchased from R&D systems. The human type 1 IFN neutralizing antibody mixture was purchased from PBL Assay Science. Anti-Zika mouse IgM antibody (Clone ZKA185) and an isotype control mouse IgM antibody (anti-fluorescein, clone 4-4-20) were obtained from Absolute Antibody.