The primary antibodies used were against SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 (mouse monoclonal, kindly provided by Dr. Peter W. Andrews, University of Sheffield, Sheffield, U.K.), Oct-4 (Santa Cruz Biotechnology, Santa Cruz, CA), a-fetoprotein (mouse monoclonal, 1:500; Sigma), cardiac troponin I (mouse monoclonal, 1: 50; Santa Cruz Biotechnology), vimentin (mouse monoclonal, 1:5; Hybrid Bank C1B7, Developmental Studies Hybridoma Bank, Iowa City, IA), human nestin (rabbit polyclonal, 1:2000; Nakama et al., unpublished results), musashi1 (rat monoclonal, 1:1000), neuronal nuclear antigen (NeuN; mouse monoclonal, 1:50; Chemicon, Temecula, CA), microtu-bular associated protein-2C (MAP-2C; mouse monoclonal, 1:500; Chem-icon), glial fibrillary acidic protein (GFAP; mouse monoclonal, 1:100; Chemicon), choline acetyltransferase (ChAT; sheep polyclonal, 1:100; Chemicon), and TH (sheep polyclonal, 1:100; Chemicon). buy generic plavix
Secondary antibodies conjugated with fluorescein isothiocyanate (green), Cy5 (red), Texas Red (red), and AMCA (aminomethylcoumarin ultraviolet-purple) were purchased from Jackson Laboratories (West Grove, PA), and those conjugated with Alexa Fluor 488 (green) were from Molecular Probes. Images were obtained from a Leica confocal system (Leica, Heidelberg, Germany) at an original magnification of 20 X or 40 X with individual filter sets for each channel. Fluorescent cells, visualized using confocal microscopy, were quantified in at least five nonoverlapping fields in each sample. The total number of cells in each field was determined by counting DAPI-positive cell nuclei. Negative controls for each fluorophore-conjugated secondary antibody, carried out without the addition of the primary antibody, detected no nonspecific binding of the secondary antibodies. The expression of alkaline phosphatase in monkey ES cells was detected following fixation of cells with 100% ethanol using a Vector blue kit (Vector Laboratories) according to the manufacturer’s instructions.
Results are expressed as the mean ± SEM. Differences were evaluated by Student f-tests. Significance was set at P < 0.05.