Nestin

Immunocytological analysis of iPSCs and embryoid bodies was as described previously [30 (link)]. Neural rosettes, NPs and neurons were fixed with 4% paraformaldehyde and stained with Pax6 (Covance), Nestin (Covance), MAP2 (AbCam) and β-tubulin III (TUJ1 clone; Covance) antibodies using standard methods.

At 3 or 4 days PI, differentiated cultures in the chamber slides were fixed in 4% para-formaldehyde and washed three times in PBS. Fixed cells were permeabilized with 0.25% TritonX-100 in PBS, washed three times, blocked with 10% normal goat serum (NGS) and immunostained using the following antibodies: Nestin (Covance Inc.; Cat# PRB-315C) at 1∶1000, neuronal class III β-tubulin (Covance Inc.; Cat# PRB-435P) at 1∶1000, GFAP (Sigma-Aldrich Co.; Cat# G 9269) at 1∶1000, MBP (Chemicon Inc.; Cat# AB980) at 1∶1000, Viral 3A protein [51] (link) at 1∶100. Primary antibodies were diluted in 2% Normal Goat Serum (NGS) in PBS (150–200 µl per slide) in humidified chamber and incubated overnight. Slides were washed with PBS for 5 min (3×). Secondary antibodies (at 1∶1000) conjugated to Alexa-Fluor-647 (Invitrogen; Cat# A21245), were diluted with 2% NGS in PBS (150–200 µl per slide) and incubated overnight. Following incubation with the secondary antibody, slides were washed with PBS for 5 min (3×), and mounted in Vectashield with DAPI. Three to five representative images of the cultures were taken for each sampling time point at multiple magnifications.

Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min and washed twice with phosphate buffered saline (PBS, Sigma-Aldrich). After membrane permeabilization with 0.3% Triton-100 (USB, Cleveland, OH, USA), cellular antigens were first blocked with 5% horse serum (Invitrogen) and then stained with primary antibodies at 4°C overnight. The primary antibodies used in this study included Oct4 (1∶500; Santa Cruz Biotechnology, Dallas, TA, USA), Sox1 (1∶200, Santa Cruz Biotechnology), Pax6 (1∶100, Covance, Princeton, NJ, USA), nestin (1∶500, Covance), βIII-tubulin (TuJ1, 1∶500, Covance), glial fibrillary acidic protein (GFAP, 1∶500, Millipore), GABA (1∶500, Millipore-Chemicon), glutamate (1∶500, Sigma-Aldrich), tyrosine hydroxylase (TH, 1∶100, Covance) and serotonin (1∶1000, Sigma-Aldrich). JEV infection was determined by an anti-JEV NS1 monoclonal antibody, a gift from Dr. Yi-Ling Lin at Academia Sinica in Taiwan. Cell nuclei were stained with diamidino-2-phenylindole (DAPI, Invitrogen). Fluorescent cell images were obtained using an upright microscope (Eclipse TE2000-S and 80i, Nikon, Tokyo, Japan) or a confocal microscope (LSM 510, Carl Zeiss, Oberkochen, Germany). The population ratios of specific protein expressing cells were estimated by manual counting with AlphaEase FC software (Alpha Innotech, San Leandro, CA, USA).