Battisti WP, Wang J, Bozek K, Murray M

Battisti WP, Wang J, Bozek K, Murray M. 1993; Taylor et al., 1993) but not for developing mouse hippocampal neurons, chick dorsal root ganglia, and isolated retinal cells of chicken (Rathjen et al., 1991; Taylor et al., 1993; Lochter et al., 1994, 1995; Lochter and SJA6017 Schachner, 1997). Consistent with an inhibitory function for optic axons, tenascin-R is not present during developmental axonal growth in the optic nerve of mice but is present in the adult (Bartsch et al., 1993). Because inhibition of axonal growth by tenascin-R Rabbit Polyclonal to LMTK3 depends on type and possibly also on developmental stage of neurons (Bates and Meyer, 1997), it is critical to directly demonstrate an inhibitory function for the cell type of interest at a specific developmental stage, in this case retinal ganglion cells of adultand inhibits optic fiber growthwere taken from our breeding colony. Animals were kept at a 12 hr light/dark cycle. Larvae were fed brine shrimp, and adults were fed beef heart. Developmental stages investigated were early larval (stages 33C34), midlarval (stages 46C48), metamorphic (stages 53C55), and adult (6C8 cm body length). Staging was carried out according to the method of Gallien and Durocher (1957). Before surgery or killing by transcardial perfusion or decapitation, animals were usually deeply anesthetized in 0.1% aminobenzoic acid ethylmethylesther (MS222; Sigma, St. Louis, MO) in PBS, pH 7.2, for 5C15 min, and the depth of anesthesia was tested by tail pinch. Proteins In vitro Bovine serum albumin (BSA), tissue culture grade, was purchased from Sigma. Tenascin-R was isolated from adult mouse brains as explained previously (Pesheva et al., 1989). The generation of glutathione Monoclonal antibodies 596 and 597 to tenascin-R (Pesheva et al., 1989), and 513 to MAG (Becker et al., 1995) and polyclonal antisera to tenascin-R (Bartsch et al., 1993) and tenascin-C (Becker et al., 1995) have previously been explained. Glial fibrillary acidic protein antibody G-A-5 was purchased from Sigma. The neurofilament antibody RT97 and the neurofilament-associated protein antibody 3A10 developed by John Solid wood (RT97) and Thomas Jessel and Jane Dodd (3A10) were obtained from the Developmental Studies Hybridoma Bank managed by the University or college of Iowa (Iowa City, IA) under contract No1-HD-7-3263 from your National Institute of Child Health and Human Development. Antibody MG5 to SJA6017 the neuronal 180 kDa isoform of neural cell adhesion molecule (NCAM) was a gift from Dr. R. Gerardy-Schahn (Medizinische Hochschule Hannover, Hannover, Germany). Western blot?analysis Cross-reactivity of antibodies 596 and 597 withtenascin-R was determined by Western blot analysis as described earlier (Becker et al., 1995), with the exception that bands of immunoreactivity were visualized using an alkaline phosphatase-coupled secondary antibody with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates. Some lanes were subsequently washed in 62.5 mm Tris-HCl, 2% SDS, and 100 mm -mercaptoethanol and immunostained with a polyclonal tenascin-C antibody to exclude cross-reactivity of antibodies 596 and 597 with tenascin-C. Antibody binding was detected with an HRP-coupled anti-rabbit secondary antibody (Dianova, Hamburg, Germany) and visualized with a chemiluminescent substrate (Amersham) according to the suppliers instructions. Blots were uncovered on Eastman Kodak (Rochester, NY) X-O-MAT film for 30C90 sec. Optic nerve?lesions Optic nerve lesions in adult salamanders were performed from a ventral approach as previously described (Becker et al., 1993, 1995). Briefly, holes were drilled into the roof of the mouth of deeply anesthetized salamanders with a dental drill to expose one or both optic nerves just outside the brain case, at a distance SJA6017 of 1 1.5C1.9 mm from your chiasm. For most experiments the nerve was then crushed with Dumont number 4 4 forceps. Only for retrograde labeling experiments was the nerve slice with a pair of microscissors (observe SJA6017 below). The wound was sealed with dental cement, and the animals were revived in tap water. Immunohistochemistry Immunocytochemistry was performed as previously explained (Becker et al.,.