1. The next day, mice were inoculated subcutaneously (via footpad) with 10 3 592 PFU of mouse-adapted ZIKV-Dak-41525;Gorman;mouse IFNAR1 blocking antibody (MAR1-5A3, from 591 Leinco Technologies),2018
2. RNA Isolation and Real-Time Quantitative RT-PCR
3. RNA 598 was reverse transcribed using a SuperScript VILO cDNA synthesis kit (ThermoFisher) according to 599 manufacturer's protocol. cDNA was then used as a template in TaqMan-PCR reactions per 600 manufacturer's instructions (Applied Biosystems) to quantify mRNA specific for IFN? (assay ID: 601 Hs01077958_s1), housekeeping gene HPRT (assay ID: Hs01003267_m1), Ajuba (assay ID: 602 Hs00262750_m1) and LIMD1 (assay ID: Hs01040528_m1). Reactions for Real-time RT-PCR were 603 set up in triplicate, cycled and data was collected on the Applied Biosystems GeneAmp 9500 , and indicated tissues were collected. Organs were individually weighed, homogenized, and 620 prepared as 10% (w/v) suspensions in DMEM/2% FBS/Pen/Strep;Total RNA was isolated from cells using RNeasy kit with genomic DNA elimination (QIAGEN)
4. Thermo Fisher 626 Scientific) overnight. To fix, cells were washed with PBS and subsequently fixed with 4% 627 paraformaldehyde for 10 min. Cells were permeabilized with 0.1% Triton X-100 for 5 min at RT, 628 and incubated with blocking solution (PBS, 0.5% BSA, 1% goat serum) for an additional 30 min. 629 Cells were then incubated with primary antibody overnight at 4�C (or 2 hours at RT), washed 630 three times with PBS and further incubated with secondary antibody (in blocking buffer) 631 conjugated to Alexa 488, 594 or 647 (Thermo Fisher Scientific) for 1 h. Slides were washed three 632 times with PBS and once with miliQ water, and mounted onto glass coverslips using Prolong Gold This;000 cells were seeded onto each well of 4 well Lab-Tek II chamber slides