Dylight 488 conjugated goat anti rabbit secondary antibody

Seeded NSCs were washed with PBS and fixed with ice-cold 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 in TBS for 5 min and blocked with 2% FBS in TBS for 30 min at room temperature. The cells were incubated with primary antibodies: rabbit anti-sox2 (1:200, cell signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-β-tubulin III (1:200, Millipore, MA, USA), rabbit anti-GFAP (1:200, Stem Cell Technologies, BC, CA), rabbit anti-phosphorylated IRF3 (Ser396) (1:200, cell signaling, CA, USA), or mouse anti-ZIKV E protein (1:500, GeneTex, CA, USA) at 4 °C overnight. The cells were then washed three times with TBS and incubated with Dylight 594-conjugated donkey anti-mouse secondary antibody or Dylight 488-conjugated goat anti-rabbit secondary antibody (1:1000, Jackson ImmunoResearch Laboratories, PA, USA) for 1 h at room temperature. The cells were then washed three times with TBS, and the cell nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, MO, USA). The images were collected with a fluorescence microscope (Olympus BX51, Olympus, Tokyo, JP).

Human NSCs and differentiated neuronal cells were seeded on chamber slides. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.5% Triton X-100 in TBS for 5 min at room temperature. TBS with 2% FBS was used for blocking. The cells were incubated with the following primary antibodies at 4 °C overnight: rabbit anti-SOX2 (1:200, Cell Signaling, CA, USA), mouse anti-nestin (1:200, Millipore, MA, USA), rabbit anti-MAP2 (1:200, Millipore, MA, USA), mouse anti-Tuj1 (anti-neuron-specific class III β-tubulin, 1:200, Millipore, MA, USA), mouse anti-GAD67 (1:200, Millipore, MA, USA), rabbit anti-active caspase 3 (1:200, Millipore, MA, USA), rabbit anti-LC3A/B (1:200, Cell Signaling, CA, USA), mouse anti-EV-A71 3D (1:500, Genetex, CA, USA) or rabbit anti-EV-A71 3A (1:500). The cells were then washed with TBS and incubated with the following secondary antibodies for 1 h at room temperature: DyLight 488-conjugated goat anti-rabbit secondary antibody or DyLight 594-conjugated donkey anti-mouse secondary antibody (1:1,000, Jackson ImmunoResearch Laboratories, Pennsylvania, USA). The cells were then washed with TBS, and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, MO, USA). Images were collected with an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan) and an LSM 510 microscope (Zeiss, Jena, Germany).