An example of a potential soluble factor is agrin, which is an extracellular matrix protein expressed by Schwann cells as well as other cells, including motor neurons (Ruegg et al., 1992). sodium channel to cluster sites containing cell adhesion molecules, and elaboration of MAG-staining Schwann cell processes adjacent to these cluster sites. Formation of the mature node of Ranvier results from the fusion of asynchronously formed pairs of clusters associated with MAG-positive Schwann cells flanking the site of presumed node formation. Studies with the hypomyelinating mutant mousedemonstrate that the elaboration of compact myelin is not required for the formation of these clustered nodal intermediates. Clustering of neurofascin and NrCAM precedes redistribution of ankyrinG 480/270 kDa and the voltage-dependent sodium channel, suggesting that the adhesion molecules define the initial site for subsequent assembly of ankyrin and the voltage-dependent sodium channel. Antibodies against the common tail region of rat ankyrinG 480 and 270 kDa were raised in chickens for double-labeling studies. Chickens were immunized with a purified recombinant polypeptide corresponding to the rat equivalent of residues 1821C2337 of the human ankyrinGsequence. This polypeptide was produced by expression in bacteria of the rat cDNA using the pGemex expression vector (Promega, Madison, WI), resulting in a fusion product with the viral gene 10 protein. Antibodies were initially purified from chicken egg yolk using an Egg Yolk Purification Kit (Pharmacia Biotech, Piscataway, NJ) and affinity-purified against purified Buspirone HCl recombinant polypeptide immobilized on Sepharose CL-6B (Pharmacia) after the previous depletion of antibodies using immobilized gene 10 polypeptide. Antibodies against neurofascin and NrCAM were generated in rabbits using purified recombinant FNIII domains (residues 581C1020 of rat neurofascin and residues 583C1018 of rat NrCAM) from these molecules as antigens. These Buspirone HCl recombinant domains were prepared in bacteria as above. Affinity-purified antibodies were then prepared from sera by purification against immobilized native 186 kDa neurofascin and NrCAM. Native proteins were purified from detergent extracts of adult rat brain membranes as described previously (Davis et al., 1993). Antibodies against neurofascin did Rabbit monoclonal to IgG (H+L)(HRPO) not cross-react with purified native NrCAM by immunoblot analysis and vice versa (Davis et al., 1996). Antibodies against the voltage-dependent sodium channel were prepared by immunization of rabbits with four multiple-antigen peptides corresponding to residues 440C453, 482C492, 547C561, and 582C600 of the I subunit (Noda et al., 1986). These sequences are relatively conserved in the three subunits expressed in the rat brain and are in an area of the molecule proposed to be a cytoplasmic linker between the I and II transmembrane pore-forming units. An area of Buspirone HCl the rat I Buspirone HCl subunit (corresponding to nucleotides 1292C2215) encompassing these peptide sequences was cloned from a rat brain library using PCR. The cDNA was subcloned into the pGemex expression vector, expressed in bacteria, and the purified recombinant polypeptide was immobilized and used in the affinity purification of the peptide antisera, as described above. Preparation of affinity-purified antibodies against brain spectrin has been described previously (Davis and Bennett, 1983). Antibodies against the MAG were purchased from Boehringer Mannheim (Indianapolis, IN). Gel samples of adult rat brain membranes were prepared as described previously (Kordeli et al., 1995) and fractionated on 3.5C17% exponential gradient gels before transfer to nitrocellulose (Davis and Bennett, 1983). Bound antibodies were visualized Buspirone HCl using 125I-labeled protein A and autoradiography (Davis and Bennett, 1983). Immunocytochemistry of frozen sections from rodent sciatic nerves was performed essentially as described (Kordeli et al., 1990). Animals of various ages were killed, and the sciatic nerves were removed by dissection. Sciatic nerves were immediately fixed in 2% paraformaldehyde either overnight at 4C or for 3 hr at 4C (for experiments involving antibodies against the voltage-dependent sodium channel) before cryopreservation in sucrose and freezing in liquid nitrogen-cooled isopentane. Primary antibodies bound on 4 m cryosections were visualized with rhodamine-labeled goat anti-rabbit Ig (Cappel, West Chester, PA) alone or in combination with fluorescein-labeled mouse monoclonal anti-chicken Ig (clone CH31, Sigma, St. Louis, MO) in double-labeling experiments. Confocal images were collected on a Zeiss LSM 410 confocal microscope, and final figures were prepared using Adobe Photoshop. RESULTS Characterization of antibodies against ankyrinG 480/270 kDa, the voltage-dependent sodium channel, neurofascin, and NrCAM Figure ?Figure11 shows an immunoblot analysis of total adult rat brain membranes (indicate the staining of axons or bundles of axons emanating from the dorsal roots. show a single bundle of axons at 2 higher magnification. These results demonstrate a major.