Gfap mouse monoclonal

TUJ1 rabbit monoclonal was from Covance (Princeton, NJ, USA product number MRB-435P). GFAP mouse monoclonal was from Sigma-Aldrich (Oakville ON Canada, product number G3893). Nestin mouse monoclonal antibody (product number MAB1259), recombinant BMP4 and recombinant TGFβ were from R&D Systems (Minneapolis MN, USA). p21 mouse monoclonal antibody, pSMAD2 rabbit monoclonal antibody and SMAD2 rabbit monoclonal antibody were from Cell Signaling Technology (Danvers MA, USA, product numbers DCS60, 138D4 and D43B4). PML mouse monoclonal antibody was from Santa Cruz Biotechnology (Santa Cruz CA, USA, product number PG-M3). LSKL peptide was from AnaSpec (Fremont CA, USA). U0126 was from Tocris Bioscience (Bristol UK).

Primary neurospheres were dissociated and plated on cover slips coated with 15 mg/ml poly-l-ornithyine (Sigma). Cells were grown for 2 days in a DMEM/F-12 medium containing EGF (20 ng/ml), FGF (20 ng/ml), and 2% FBS and then transferred to a differentiating medium (EGF and FGF free plus 1% fetal bovine serum) for 5 days. Every 2 days, half of the medium was replenished with fresh differentiating medium. ELN 1 nM was administered on alternate days throughout the differentiation period (6 days) starting from the first day. Differentiated cells were fixed (see above) and incubated with anti-glial fibrillary acidic protein (1:400; GFAP mouse monoclonal, Sigma) and anti-β-III tubulin (1:100; rabbit polyclonal, Sigma), as primary antibodies, and with anti-mouse FITC-conjugated (1:200; Sigma) and anti-rabbit Cy3-conjugated (1:200; Jackson Laboratories), as secondary antibodies. Samples were counterstained with Hoechst-33258. Cells were counted in five different fields of each cover slip. Number of positive cells for each antibody was referred to the total number of Hoechst-stained nuclei. Evaluation of total neurite length of cells differentiated into neurons was performed by using the image analysis system Image-Pro Plus (Media Cybernetics, Rockville, MD, USA).