Immunocytochemical staining was performed following the protocol described before (Zhang et al., 2001 (link)). Antibodies used included rabbit anti-Oct4 (1:200, Abcam, Cambridge, MA, http://www.abcam.com ), goat anti-Sox2 (1:1,000, R&D, Minneapolis, MN, http://www.rndsystems.com/ ), mouse anti-Pax6 (1:5,000, DSHB, Iowa City, IA, http://dshb.biology.uiowa.edu/ ), rabbit anti Olig2 (1:500, Chemicon & Millipore, Billerica, MA, http://www.millipore.com ), mouse anti Hb9 (1:50, DSHB), goat anti ChAT (1:300, Chemicon & Millipore), Rabbit GFAP (1:5000, DAKO, Carpinteria, CA, http://www.dako.com ), rat anti MAP2 (1:1000, Chemicon & Millipore), mouse anti hGFAP (1:500, Stem Cells Inc, Newark, CA, http://www.stemcellsinc.com/ ).
Images were collected with a Nikon TE600 fluorescence microscope (Nikon Instruments, Melville, NY) or a Nikon C1 laser-scanning confocal microscope (Nikon, Tokyo, Japan). The populations of MAP2/GFP, hGFAP /GFP positive cells in the spinal cord were counted in fields chosen by an automated stage movement operated by Stereo Investigator software (MicroBrightField Inc.) or using Z-section images analyzed using image J software. GFP positive neurons or GFP positive astrocytes were counted every six sections as described (Ma et al., 2012). For cell quantification in vitro, 150-200 neurons were counted from 3 repeated experiments. Data are presented as mean ± SEM.
Images were collected with a Nikon TE600 fluorescence microscope (Nikon Instruments, Melville, NY) or a Nikon C1 laser-scanning confocal microscope (Nikon, Tokyo, Japan). The populations of MAP2/GFP, hGFAP /GFP positive cells in the spinal cord were counted in fields chosen by an automated stage movement operated by Stereo Investigator software (MicroBrightField Inc.) or using Z-section images analyzed using image J software. GFP positive neurons or GFP positive astrocytes were counted every six sections as described (Ma et al., 2012). For cell quantification in vitro, 150-200 neurons were counted from 3 repeated experiments. Data are presented as mean ± SEM.