Cy2tm conjugated affini pure donkey anti rabbit igg

The differentiation of neuronal cells was analysed by Laser Scanning Confocal Microscopy (LSCM, Fluoview FV300, Olympus, Milan, Italy) after the immunostaining of specific neuronal markers, such as β-tubulin III, a protein associated with the cytoskeleton that is present in the soma and in all neuronal extensions; growth-associated protein-43 (GAP 43), a specific component of axonal extensions; and synaptophysin, a synaptic vesicles marker. Neurons were fixed in 4% (wt/v) paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 in PBS for 10 min and then blocked with 1% (v/v) BSA for 30 min at room temperature. Then, samples were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-βTub III (1:500, Covance, Princeton, NJ, USA), mouse anti-synaptophysin (1:400, Chemicon, Millipore, Burlington, MA, USA) and goat anti-GAP-43 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies, Cy2^TM-conjugated Affini Pure donkey anti-rabbit IgG, Cy3^TM-conjugated Affini Pure donkey anti-mouseIgG and a Cy5^TM-conjugated Affini Pure donkey anti-goat IgG (1:500, Jackson ImmunoResearch Europe Ltd., Cambridge, UK), were added for 1 h at RT. Finally, cells were counterstained with 200 ng/mL DAPI (Molecular Probes) for nuclear localization. Samples were rinsed, mounted and observed at LSCM.