Cy3 conjugated anti mouse secondary antibody

For proliferation analysis, neurospheres were dissociated in a single cell suspension and plated onto poly-l-ornithine-coated 24-well chamber slides at a density of 3 × 10⁴ cells per well in a DMEM/F-12 medium containing EGF (20 ng/ml), FGF (20 ng/ml) and 2% FBS. Cells were treated with ELN 1 nM and, in some experiments, with 10 μg/ml cyclopamine hydrate (Sigma) for 24 h. Cells were then treated with BrdU for additional 6 h, paraformaldehyde fixed and stained with a mouse anti-5-bromo-2-deoxyuridine (BrdU) monoclonal antibody (1:100; Roche Applied Science) and a Cy3-conjugated anti-mouse secondary antibody (1:200; Sigma). Samples were counterstained with Hoechst-33258. Digital images were captured using an Eclipse TE 2000-S microscope and the NIS-Elements AR software (Nikon).

Roscovitine-treated and untreated neurons were washed with 1× Phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (Invitrogen Life Technologies), stained with β-tubulin III antibody (1:200 dilution; Sigma-Aldrich Co.) in 1× PBS containing 10% goat serum and 0.3% Triton X-100 and Cy3-conjugated anti-mouse secondary antibody (1:200 dilution; Sigma-Aldrich Co.) in 1× PBS containing 10% goat serum and 0.3% Triton X-100. Images were captured under a Zeiss fluorescence microscope. Neurite length was analyzed by the MetaXpress software and by an observer blinded to their condition (Molecular Devices, Sunnyvale, CA, USA). Between 40 and 60 neurons were analyzed per condition. Three to four rats per condition were routinely used. Graph Pad Prism was used for statistical analysis. Student’s t-tests were carried out with the statistical significance set at p ≤ 0.05.