Anti mushashi1

Cells were fixed using paraformaldehyde, 4% in PBS, for 15 minutes at room temperature. After washing, the cells were permeabilized with 0.1% Triton X-100 (Promega) diluted in PBS for 15 minutes. After blocking with 2% of BSA (Sigma-Aldrich) for 4 hours, primary antibodies directed against the following were added: anti-ZIKV (polyclonal mouse, Institute Pasteur in Dakar, 1:80), anti-Flavivirus D1-4G2-4-15 (polyclonal mouse, Millipore, 1:100), 1:50, anti-MAP2 (chicken, Abcam ab5392, 1:200), anti-cleaved caspase-3 (rabbit, Cell Signaling #9661, 1:400), anti-Sox2 (mouse, Abcam ab97959), anti-GFAP (rabbit, Abcam, 1:500) and anti-Mushashi1 (rabbit, Abcam, 1:1000) (Supplementary Table 2). The cells were incubated overnight at 4°C. Secondary antibodies were added for a one-hour incubation at room temperature, being the following ones: anti-mouse Alexa Fluor 488, anti-chicken Alexa Fluor 647, anti-rat Alexa Fluor 555 and anti-rabbit Alexa Fluor 555 (Invitrogen). The nuclei were stained with DAPI (Invitrogen, 1:10,000) diluted in a PBS 1x solution for 5 minutes and mounted with DPX (Sigma). Images were acquired with Nikon Eclipse 80i. Analysis of data was performed using software NIS Elements 3.22 (Tokyo, Japan).