βiii tubulin tuj1

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).

Cells seeded in 4-well plates were fixed with 4% paraformaldehyde (Sigma-Aldrich) and washed twice with phosphate-buffered saline (PBS). The cells were permeabilized with 0.3% Triton-100 for 10 min and then treated with 5% horse serum. Primary antibodies were added and incubated at 4°C overnight. The primary antibodies used in this study included those against Sox-1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), Pax-6 (1:100, Covance, Princeton, NJ, USA), Nestin (1:500, Covance), N-cadherin (1:500, Santa Cruz Biotechnology), Forse-1 (1:50, Development Studies Hybridoma Bank, DSHB, IA, USA), βIII-tubulin (TuJ1, 1:500, Covance), MAP2 (1:1000, Merck Millipore), phospho-PHF-Tau pSer²⁰²/Thr²⁰⁵ (AT8, 1:200, Thermo Scientific, Rockford, IL, USA), β-Amyloid 1-42 (Aβ42, 1:500, Merck Millipore) and β-catenin (1:500, BD). The cell nuclei were stained with diamidino-2-phenylindole (DAPI). Fluorescence images were captured using an upright microscope (Eclipse TE2000-S and 80i, Nikon, Tokyo, Japan) or a confocal microscope (LSM 510, Carl Zeiss, Oberkochen, Germany).