Embryoinic stem cells cultured and embryoid bodies (EB) treated to retonic acid to develop neurons.
Immunocytochemistry EBs processed for immunohistochemistry with antibodies noted below.Images were obtained using a BioRad confocal microscope.
Antibody Reagents:Rabbit aHB9, aOlig2, aNkx2.2, aLhx3, aDbx1 (Pierani, 1999), aIrx3, aGFP (1:2000, Molecular Probes), aSox1 (1:500 [Pevny et al., 1998]), aOtx2 (1:1000 [Mallamaci et al., 1996]), aHoxC5 (Liu et al., 2001), and aChx10; mouse aPax7, aPax6, aHB9, aLim1, aNkx6.1, aNkx2.2, aEn1, aLim2 (mouse specific), aNCAM (mouse specific), aSC1 (chick specific), aNeuN (1:500, Chemicon), aTuj1 (1:2000, BABCO), aSyb (mouse specific, 1:1000, Synaptic Systems), aSyn (1:1000, Synaptic Systems), and aNeurofilament; guinea pig a-Isl1, anti-Olig2, and anti-Hoxc6 (Liu et al., 2001); goat aChAT (1:200, Chemicon) and aVAChT (mouse specific, 1:1000, Chemicon) All antibodies cited in Novitch et al. (2001) when not otherwise specified. FITC-, Cy3-, and Cy5-conjugated secondary antibodies were used (Jackson Immunoresearch).
transgenic mouse line developed (HBG3)
Facs analysis done on HBG3 mouse line showed 95% motor neurons.
Transplantation: motor neuron axon development studied in chick embryos with rodamine conjugated bungarotoxin or other relevent antibodies.