Foxg1

Staining was performed on cells fixed with ice-cold 4% paraformaldehyde and subsequently permeabilized in blocking solution (1× phosphate-buffered saline pH 7.4, 1% bovine serum albumin, 0.1% Triton X-100). Primary antibodies against FOXG1 (1:100; abcam, Cambridge, United Kingdom), NESTIN (1:300; R&D systems, MN, United States), MAP2 (1:5,000; abcam, Cambridge, United Kingdom), GABA (1:1,000; Sigma, MO, United States), GAD1 (1:100, Millipore, MA, United States) and SST (1:100, Millipore, MA, United States) were used for immunostaining and quantification. Primary antibodies were allowed to bind overnight separately or in appropriate combinations at 4°C. After washing three times in 1×TBS, 0.05% Tween, the secondary antibodies donkey anti-goat IgG AlexaFluor 633, donkey anti-rabbit IgG AlexaFluor 568 or donkey anti-mouse IgG AlexaFluor 488 (1:1,000; ThermoFisher Scientific, Waltham, MA, United States) were applied alone or in appropriate combinations for 1.5 h at room temperature in the dark. Visualization was performed on a Zeiss 510 confocal microscope (Carl Zeiss Microscopy, Jena, Germany) using Zen 2009 imaging software.

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 10 min, and blocked with 3% bovine serum albumin for 30 min at room temperature. After blocking, cells were incubated overnight at 4 °C with primary antibodies. Primary antibodies are NANOG, TRA-1-81 (1:250, Stem cells); KRT8, KRT18, KRT19, AP-2γ, FOXG1 (1:200, Abcam); ALDH1A3, AP-2α, AFP (1: 200, Santa Cruz); Brachyury (T) (1:50, Novus); P63 (1:100, Genetex); p-SMAD1/5/9 (1:200, Cell Signaling); CDH1, Desmoplakin (1:500, BD Biosciences). Alexa 594-conjugated secondary antibody (1:400, red, Molecular Probes, Eugene, OR) or an Alexa 488-conjugated secondary antibody (1:400, green, Molecular Probes) was used to visualize the staining. Following three washes with PBS, slides were mounted with the VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA). Prior to mounting, slides were incubated with 2 μM 4′,6-diamidino-2-phenylindole (DAPI) fluorescence (Molecular Probes) for 10 min at 37 °C to stain the nuclei. The fluorescence images were taken using the EVOS FL Auto Cell Imaging System fluorescence microscope (ThermoFisher Scientific, NY, USA).

Coverslip cultures were fixed in 4% paraformaldehyde for 10 min at room temperature. After adequate washing with PBS, cells were incubated in a blocking buffer (10% donkey serum plus 0.2% Triton X-100 in PBS) for 60 min at room temperature followed by primary antibody incubation at 4 ^oC overnight. On the next day, coverslips were washed with PBS and stained with the fluorescently conjugated secondary antibodies (1:1000, Jackson, West Grove, PA). Nuclei were counterstained with Hoechst 33258. Coverslips were visualized with Leica TSC SP5 (Leica Microsystems, Bensheim, Germany) confocal laser-scanning microscope. Antibodies used in this study included Pax6 (1:1000, rabbit IgG, Covance), Nkx2.1 (1:400, mouse IgG, Chemicon), FoxG1 (1:1000, rabbit IgG, Abcam), Sox1 (1:500, goat IgG, R&D), Sox2 (1:1000, goat IgG, R&D), Oct4 (1:1000, mouse lgG, Santa Cruz), HoxB4 (1:50, mouse IgG, DSHB), Nestin (1:500, mouse IgG, millipore), Map2 (1:10,000, chicken IgY, abcam), Tuj1 (1:5000, mouse IgG, sigma), cleaved caspase 3 (1:500, rabbit IgG, CST), BrdU (1:200, rat IgG, Abcam), Ki67 (1:500, rabbit IgG, Abcam), pH3 (1:500, rabbit IgG, CST) and ZIKV (1:500, mouse anti-flavivirus group antigen antibody, millipore). Light images were visualized with Leica DMI3000 (Leica Microsystems, Bensheim, Germany) microscope and organoid size was measured with Leica application Suite softwares.