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The solvent was evaporated at low than 40°C and the remaining solution was spray-dried to obtain Salacia extract. One week after injection, labeled animals were perfused with 4% phosphate-buffered (0.1 m, pH 7.4) paraformaldehyde solution. The labeled brains were incubated at 37°C in 30% sucrose-4% paraformaldehyde solution for 2 months and then sectioned and analyzed as described above. The sections were washed twice in RNase buffer (10 mm Tris-HCl, pH 7.5, 0.5 m NaCl, and 5 mm EDTA) for 20 min each and incubated for 30 min at 37°C in the same buffer containing 20 μg/ml RNase A. Sections were then rinsed in the RNase buffer for 20 min at 37°C. Finally, the sections were washed in 50% formamide, 2× SSC at 65°C for 30 min and in 2× SSC and 0.1× SSC at room temperature for 15 min, respectively. After they were blocked in TBS containing 0.2% Triton X-100 (TBST) and 1% gelatin hydrolysate (DGF, Eberbach, Germany), the membranes were incubated with anti-rMAL antibodies (2.5 μg/ml), anti-PLP antibody (Immunodiagnostics Inc., Bedford, MA) (diluted 1:500), or O1 or O4 hybridoma supernatant (Sommer and Schachner, 1981) (gift from Dr. M. Schachner, University of Hamburg) diluted 1:10 and 1:5, respectively, overnight at 4°C. After they were washed in TBST, signal detection was performed by incubation with horseradish peroxidase-labeled (Amersham, Buckinghamshire, UK) or alkaline phosphatase-labeled secondary antibodies (Jackson) using chemiluminescent reagents (Amersham) or BCIP/NBT as a substrate.

The cocultures were maintained for 48 hr, fixed with 4% paraformaldehyde in PBS, and then immunocytochemically stained with anti-neuron-specific enolase (for rat neurons) (Gao et al., 1996) or anti-neurofilament H (for mouse neurons) antibodies using Vectastain ABC kit (Vector Laboratories, Burlingame, CA). In situ hybridization. Embryonic day (E) 18 CD-1 mouse embryos and early postnatal (P) mice (P5 and P7) (Charles River Laboratories, Wilmington, MA) were used in in situ hybridization experiments. Mouse ephrin-B3 was cloned into a retroviral vector pLIG (Lillien, 1995), which contains a β-galactosidase gene fused to an aminoglycoside phosphotransferase for G418 resistance. Primary neuron culture. The hippocampi from E18 rat or E16 mouse embryos were dissected in PBS, dissociated into a single-cell suspension by mechanically passing through polished glass pipettes, and counted under a microscope. 10,000 molecular weight; Molecular Probes (Eugene, OR), D-1817) suspension (10% in saline) was stereotaxically injected into medial or lateral hippocampus with iontophoresis (8 μA, positive alternating current for 30 min) under surgical microscope. Crystal DiI (Molecular Probes, D-3911) was inserted into the lateral hippocampal regions. Hippocampal neuron aggregates of similar size on control or ephrin-B3-expressing cells were selected for quantification. For tracing adult hippocampal axons, mice were anesthetized with Nembutal (50 mg/kg body weight, i.p.).

The mothers were anesthetized with nembutal (50 mg/kg body weight, i.p.), the embryos were dissected and decapitated, and the heads were frozen on dry-ice powder. Quantification of cell aggregation and axon bundle thickness. Sagittal sections of 12 μm thickness were cut with a cryostat at -23°C and mounted on VWR Scientific superfrost slides. To hybridize with riboprobes, the sections were treated with proteinase K (40 ng/ml), refixed with 4% paraformaldehyde, immersed in triethanolamine (50 mm) in acetic anhydride solutions (100 mm) for 10 min, and dehydrated. Coronal sections (100 μm) were cut with a vibratome, collected in phosphate buffer (0.1 m, pH 7.4), and mounted on slides treated with poly-l-lysine. The sections were exposed for 2-3 weeks at 4°C, developed, and counterstained with thionin (0.25% in 10% acetic acid adjusted to pH 4 with NaOH). The sections were hybridized with the respective 35S- (2.5 × 106 cpm/ml) or digoxigenin-labeled riboprobes under stringent conditions (50% formamide, 10% dextran sulfate, 1× Denhardt's solution, 0.2 mg/ml herring sperm DNA, and 10 mm dithiothreitol) for 18-24 hr at 55°C. After hybridization, the sections were washed in 5× SSC at 65°C for 20 min, followed by 50% formamide in 2× SSC for 30 min at the same temperature.