Goat anti fzd1

N2a cells were homogenized in RIPA buffer (10 mM Tris/HCl pH 7.4, 5 mM EDTA, 1 % NP-40, 1 % sodium deoxycholate and 1 % SDS) supplemented with a protease inhibitor mixture (1 mM PMSF, 2 μg/ml of aprotinin, 1 μg/ml of pepstatin and 10 μg/ml of benzamidine) and phosphatase inhibitors (25 mM NaF, 100 mM Na₃VO₄, 1 mM EDTA and 30 μM Na₄P₂0₇). Homogenates were maintained on ice for 30 min and then centrifuged at 1000 g for 5 min (4 °C) to remove nuclei and large debris. Protein concentration on supernatants was determined using the BCA Protein Assay Kit (Pierce). Proteins were resolved in 10 % SDS/PAGE, transferred to a PVDF membrane and incubated overnight at 4 °C with primary antibodies. Primary antibodies used were: goat anti-FZD1 (R&D Systems), rabbit anti-β-actin (Cytoskeleton, Inc), mouse anti-β-catenin (Santa Cruz Biotechnology, Inc.), rabbit anti-α-tubulin (Santa Cruz Biotechnology, Inc.). The reactions were followed by incubation with peroxidase-conjugated secondary antibodies (Pierce) and developed using the ECL technique (Western Lightning Plus ECL, PerkinElmer).

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).