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Staining of all motor axons [with antibodies against neurofilaments, synaptic vesicles (SV2), and acetylated microtubules] was done similarly as described in (11). for tetanus toxin labeling, the preparation was incubated with a fluorescein isothiocyanate-conjugated recombinant tetanus toxin C fragment (Boehringer Mannheim) for 20 min (1:10 dilution).
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To measure the separation of two competing axons, we calculated a segregation index as the distance between centroids of the competing axons divided by the total length of the junction, which was measured along a line passing through the two centroids. The centroid (geometric center) of the terminal branches of each competing axon was obtained with IP Lab software (Scanalytics, Fairfax, VA). The particular shape of the junction will influence this measure to some degree. For hemicircles that approximate the shapes of competing axon terminals, values >0.39 indicate that the regions are completely segregated.
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Because of the low incidence of multiply innervated junctions after P12, we used antibody staining instead of lipophilic dyes to label competing axons at P16-17. Competing axons innervating the same junction at this age were traced several hundred micrometers back into nerve bundles to confirm that they were separate axons.
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We thank all the members of our lab for helpful discussion of this work; J. R. Sanes, R. O. Wong, and M. L. Nonet for comments on the manuscript; and S. G. Turney for technical help. This work was supported by grants from NIH and the Muscular Dystrophy Association. W.-B.G. was supported by a National Research Service Award from NIH.
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