Tuj1 clone

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.

Neurospheres and dissociated cells were incubated overnight at 4°C with primary antibodies raised against the neuroepithelial cell marker nestin (rabbit, 1:2000; a gift from Dr. R. McKay, NIH, Bethesda, MD, USA); the S-phase marker BrdU (mouse 1:250, Becton Dickinson, San Jose, CA, USA, No.347580); the general neuronal marker β-III-tubulin (TuJ1 clone, mouse, 1:1,000; Covance, Berkeley, CA, USA, No. MMS-435P; and rabbit antibody, 1:300; Abcam No. ab18207); the astrocyte marker GFAP (rabbit polyclonal, 1:1,000; Dako, Glostrup, Denmark, No. Z0334; and mouse 1:1500; Millipore No. MAB360); the oligodendrocyte marker O4 (mouse monoclonal IgM, 1:8; obtained from the culture media of O4-producing hybridoma cells kindly provided by A. Rodríguez-Peña, CSIC, Madrid, Spain; and O4, 1:150; Millipore, Temecula, CA, USA, No. MAB345); and the presynaptic vesicle protein synaptophysin (rabbit polyclonal, 1:6: Invitrogen, Camarillo, CA, USA, No. 18-0130). Antibody binding to the cells was then detected using Alexa Fluor^® 488, 594, and Texas Red^® conjugated secondary antibodies (1:500, Invitrogen) and finally, nuclei were stained with Hoechst (Sigma). Controls were performed to confirm the specificity of the primary and secondary antibodies.