Monoclonal anti gfap

Immunodetection of neuronal markers was carried out as previously described [53 (link), 54 (link)]. Primary antibodies used were: rabbit anti-doublecortin (Cell Signaling Technology Inc.), monoclonal anti-NeuN (Millipore), goat anti-FZD1 (R&D Systems), goat anti-FZD1 (LifeSpan Biosciences, Inc.), rabbit anti-SOX2 (Cell Signaling Technology Inc.), monoclonal anti-GFAP (Sigma-Aldrich), monoclonal anti-Nestin (Millipore), rabbit anti-Ki67 (Abcam). As secondary antibodies, Alexa (Molecular Probes) and DyLight (Abcam) conjugated antibodies were used. NucBlue (Life Technologies) was used as nuclear dye. Slices were mounted on gelatin-coated slides with Fluoromont-G (Electron Microscopy Sciences). Double-labeled sections were analyzed by confocal laser microscopy (Olympus FV 1000). Image analysis and z-projections were made with ImageJ software (NIH, USA).

Proteins of spinal cord homogenates were separated by SDS-PAGE and then electro-transferred onto nitrocellulose membranes. The membranes were incubated in PBS (80mM Na₂HPO₄, 20mM NaH₂PO₄, 100mM NaCl; pH 7.5) containing 0.05% Tween 20 (PBS-T) and 5% milk powder for 1.5 h at room temperature. The membranes were then washed in PBS-T and incubated overnight at 4°C in PBS-T containing 5% milk powder and primary antibody: monoclonal anti-GFAP (Sigma-Aldrich, G3893) in dilution 1:400 or monoclonal anti-actin (MP Biomedicals, 0869100) in dilution 1:500 (internal standard). After washing in PBS-T, membranes were incubated for 30 min at room temperature in PBS-T containing 5% milk powder and secondary antibody: peroxidase-conjugated anti-mouse IgG (1:5000) (Sigma-Aldrich, A2304). The membranes were then washed in PBS-T, the cross-reacted antibodies were detected using a chemiluminescence system (ECL Western Blotting System, Amersham Bioscences) and exposed to Hyperfilm ECL. The films were scanned and quantified using Image Quant TL v2005.