Rabbit polyclonal anti gfap

The following primary antibodies were used: goat polyclonal anti-AQP4 (Santa Cruz, CA, USA); goat polyclonal anti-CD31 (Santa Cruz, CA, USA); rabbit polyclonal anti-GFAP (Sigma-Aldrich, Milan, Italy); mouse monoclonal anti-GFAP (clone G-A-5, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-GAPDH (MAB 374, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-Glutamine Synthetase (MAB 302, Millipore, Merck); goat polyclonal anti-actin (Santa Cruz, CA, USA). The following secondary antibodies (all from Invitrogen, Milan, Italy) were used for immunofluorescence: donkey anti-goat Alexa Fluor488; donkey anti-mouse Alexa Fluor488; donkey anti-rabbit Alexa Fluor594; donkey anti-goat Alexa Fluor594; donkey anti-rabbit Alexa Fluor647. The following secondary antibodies (all from Santa Cruz, CA, USA) were used for Western blot:
goat anti-mouse IgG-horseradish peroxidase (HRP); goat anti-rabbit IgG-HRP; donkey anti-goat IgG-HRP.

U87 neurospheres at day 14 were trypsinized and disaggregated in order to have a single cell suspension. 4 Â 10 4 cells were plated in 60 mm tissue culture petri dishes in DMEM supplemented with 10% FBS in presence or absence of 10 mM paxilline and allowed to differentiate for 5 days. Total RNA was extracted and RT-PCR was performed to evaluate mRNA expression of the differentiation marker GFAP and the stemness markers oct-4, sox-2, and nanog. For immunofluorescence, 5 Â 10 3 cells were plated on chamber slides (Nunc Lab-Tek), fixed with 4% paraformaldehyde, permeabilized with 0.5% PBS-triton X-100, blocked 1 h with 0.2% PBS-gelatin and stained overnight at 4°C with the following primary antibodies: rabbit polyclonal anti-GFAP (dilution 1:300, Sigma-Aldrich), mouse monoclonal anti-nestin (dilution 1:300, Santa-Cruz, Dallas, TX) and mouse monoclonal anti-b-III tubulin (dilution 1:300, Sigma-Aldrich). Secondary antibodies used were: goat anti-mouse Alexa-fluor 594 conjugate and goat anti-rabbit Alexa-fluor 488 (dilution 1:1000, Life Technologies). Nuclei were counterstained with DAPI for 5 min in the dark and mowiol was used to mount coverslips on chamber slides. Images were acquired with Nikon Eclipse Ni motorized microscope system at 10Â magnification.