Anti betatubulin

The cells were fixed for 20 min in 4% paraformaldehyde in phosphate-buffered saline, pH 7.4, with phosphate-buffered saline/0.1% Triton-X containing 10% normal goat serum. The primary antibodies were anti-Nestin (1:200, rabbit, Sigma-Aldrich) for detection of undifferentiated NSCs, anti-MAP2 (1:200, rabbit, Sigma-Aldrich) for detection of mature neurons, anti-betatubulin ІІІ (1:100, rabbit, Sigma-Aldrich) for detection of immature neurons, anti-O4 (1:100, mouse, monoclonal, Sigma-Aldrich) for detection of immature oligodendrocyte and anti-GFAP (1:400, mouse, clone G-A-5, Sigma-Aldrich) for detection of astrocytes.
After washing, the cultures were secondary antibodies labeled with fluorescine iso-thiocyanate, tetra methyl rhodamyne iso-thiocyanate and Phycoerythrin, then washed, incubated with 4′, 6-diamidino2 phenylinole dihydrochcloride (1 mg ml -1 in methanol, 15 min at 37 °C and finally mounted using Fluorsave (Calbiochem; La Jolla, CA, USA)

At the end of behavioral testing, the animals were anesthetized, and the SC tissues cryoprotected in 30% sucrose were cronally sectioned, and stained with Anti-GFP (1:2000, rabbit, N-Terminal, Sigma-Aldrich) and cell type-specific markers. The following primary antibodies were used: anti-Nestin (1:200, rabbit, Sigma-Aldrich) for detection of undifferentiated NSCs; anti-MAP2 (1:200, rabbit, Sigma-Aldrich) for detection of mature neurons; anti-beta-tubulin ІІІ (1:100, rabbit, Sigma-Aldrich) for detection of immature neurons; anti-O4 (1:100, mouse, monoclonal, Sigma-Aldrich) for detection of immature oligodendrocytea; and anti-GFAP (1:400, mouse, clone G-A-5, Sigma-Aldrich) for detection of astrocytes. Fluorescine iso-thiocyanate, (anti-rabbit, anti-mouse, anti-chicken, Sigma-Aldrich), tetra methyl rhodamyne iso-thiocyanate (anti-rabbit, Sigma-Aldrich) and Phycoerythrin (anti-mouse, anti-goat, Sigma-Aldrich) were applied, and then the SC tissues were examined under a fluorescent microscope.