A 11039

The human NPCs were fixed with 4% paraformaldehyde (PFA, Synth, Diadema, SP, Brazil, 01P1005.01) for 1 h at 4 °C, permeabilized and blocked with 5% BSA solution (LGC, Sao Paulo, SP, Brazil, 9048-46-8) and 0.5% TritonX-100 (LGC, Sao Paulo, SP, Brazil, 13-1315-05) for 30′ at room temperature. Primary antibodies were diluted in blocking solution and labeled overnight at 4 °C. The following day, secondary antibodies diluted in blocking solution were used for 2 h in the dark at room temperature. Finally, DAPI (Invitrogen, Itapevi, SP, Brazil, D1306) was used to label the cell nucleus at a 1:5000 dilution in 1× PBS for 10 min in the dark at room temperature. Visualization was performed using a ZOE Biorad^® (Hercules, CA, USA) microscope. Primary antibodies: anti-ZIKV envelope protein (rabbit, GeneTex, Irvine, CA, USA, GTX133314, 1:200); anti-MAP2 (chicken, Abcam, Cambrigde, UK, ab5392, 1:200); anti-SOX2 (goat, Abcam, Cambrigde, UK, ab97959, 1:200); anti-musashi-1 (goat, Abcam, Cambrigde, UK, ab52865, 1:200); anti-nestin (mouse, Abcam, Cambrigde, UK, ab7659, 1:200). Secondary antibodies: anti-chicken 488 (Invitrogen, Waltham, MA, USA, A-11039, 1:500); anti-rabbit 555 (Abcam, Cambrigde, UK, ab150078, 1:500); anti-goat 488 (Invitrogen, Waltham, MA, USA, A32184, 1:500); anti-goat 555 (Invitrogen, Waltham, MA, USA, A21432, 1:500).

To assess brain cellular subtype markers (i.e., neurons, microglia, astrocytes, and oligodendrocytes) co-expressed with EGFP, striatal and hippocampal monkey sections were incubated simultaneously with two antibodies: a chicken polyclonal antibody against GFP (Abcam, ab13970, 1:1000) and, respectively, a mouse monoclonal antibody against NeuN (Millipore, Darmstadt, Germany, MAB377, 1:1000) or a rabbit monoclonal against human Iba1 (FUJIFILM Wako Pure Chemical Corporation, 019-19741, 1:1000) or a mix of mouse monoclonal antibodies against GFAP and S-100 (Sigma-Aldrich (clone GA5), 1:2000/Abcam, ab7852, 1:1000) or a mouse monoclonal antibody against CNPase (Abcam, ab237961, 1:1000) for one night at room temperature. Incubation with secondary antibodies was done sequentially with a goat anti-chicken AlexaFuor 488 (Abcam, A11039, 1:400) and a goat anti-mouse AlexaFluor 568 (Invitrogen, A11031, 1:400) or a goat anti-rabbit AlexaFluor 568 (Invitrogen, A11036, 1:400) 1h30 at room temperature. Sections were mounted on non-gelatinized slides and coverslipped using fluorescent mounting media without DAPI (Vector Labs). Images were acquired using a Zeiss SP5 confocal microscope.