Miner JH, Li C. for migrating axons (Kuhn et al., 1995; Halfter, 1996; Kuhn et al., 1998), and as an important guidance molecule for developing axons (Garcia-Alonso et al., 1996; Forrester and Garriga, 1997). One class of laminin receptors, the integrins, are critical in mediating laminin-induced neurite outgrowth ABC294640 (for review, see Powell and Kleinman, 1997) and are modulated depending on laminin availability (Condic and Letourneau, 1997), laminin conformation (Calof et al., 1994; Ivins et al., 1998), and developmental age (Cohen et al., 1986,1989; Hall et al., 1987; Ivins et al., 2000). This suggests dynamic interplay between laminin and integrins around the neuronal cell surface. In this study, we examine the role of the permissive cue laminin in axon guidance. Sequence analysis of the grasshopper laminin -chain demonstrates a single conserved nidogen-binding site that has been shown to be important for epithelial tissue morphogenesis in other systems (Gerl et al., 1991; Mayer et al., 1993a; Ekblom et al., 1994; Poschl et al., 1996; Kadoya et al., 1997). We show that this nidogen-binding site is usually important for axonal pathfinding and may be required for growth cone turning Embryos were dissected out of their egg cases in saline, and the amnion was removed and staged according to the method of Bentley et al. (1979). Embryos were fixed for 1 hr in 3.7% formaldehyde in PIPES, EGTA, and MgSO4. Embryos were blocked for 1 hr at 4C in either PBT and 5% normal goat serum or PBT and 5% normal donkey serum, depending of the host of the secondary antibody. Primary antibodies (see below) were incubated overnight at 4C, followed by several washes in PBS Mouse monoclonal to Histone 3.1. Histones are the structural scaffold for the organization of nuclear DNA into chromatin. Four core histones, H2A,H2B,H3 and H4 are the major components of nucleosome which is the primary building block of chromatin. The histone proteins play essential structural and functional roles in the transition between active and inactive chromatin states. Histone 3.1, an H3 variant that has thus far only been found in mammals, is replication dependent and is associated with tene activation and gene silencing. supplemented with 0.1% bovine serum albumin and 0.1% Triton X-100, pH 7.2 (PBT) and secondary antibody incubation at 1:250 in PBT for 1 hr at room temperature. Embryos were again washed in PBT and mounted in Slowfade antifade (Molecular Probes, Eugene, OR). Primary antibody concentrations were as follows: goat anti-HRP, 1:500; rabbit anti-HRP, 1:500; rabbit anti-laminin -chain, 1:500; rabbit anti-semaphorin 2a, 1:250; mouse anti-semaphorin 1a, 1:1; rabbit anti–integrin (against the full-length protein), 1:20; and rabbit anti- integrin (against the intracellular domain name), 1:50. Rabbit anti–integrin antibodies were courtesy of Salvatore Carbonetto (McGill University, Montral, Qubec, Canada). Goat anti-HRP and rabbit anti-HRP were from Jackson ImmunoResearch (West Grove, PA), as were the secondary antibodies used ABC294640 in this study (FITC-conjugated donkey anti-goat, FITC-conjugated donkey anti-rabbit, Cy3-conjugated goat anti-mouse, and FITC-conjugated goat anti-rabbit). For double labeling, primary antibodies were incubated together with embryos overnight at 4C. Secondary antibodies were also incubated together for 1 hr at room temperature. For integrin immunofluorescence, embryos were immobilized on glass coverslips previously coated in 5 mg/ml poly-l-lysine and filleted along the proximalCdistal ABC294640 axis of the limb (O’Connor et al., 1990). Filleted limbs were rolled open to expose the ventral epithelium, made up of the Tibial (Ti1) neurons. Embryos were fixed and stained with anti-1-integrin antibodies. Confocal immunofluorescent images were collected on a ABC294640 Nikon Optiphot-2 microscope using an MRC 600 confocal system (Bio-Rad, Hercules, CA) equipped with a kryptonCargon laser. The images collected from the confocal microscope were captured in a 768 512 pixel field of view with the optical sections collected at 0.8 mm intervals. The confocal images were composed of 100C150 optical sections for each embryo. Data collected from the confocal microscope were analyzed in NIH Image 1.61, and Adobe (Mountain View, CA) Photoshop 4.0 was used for presentation. Confocal microscopy was conducted at the Electron Microscopy facility at the University of British Columbia. The IgG fraction of immune and preimmune sera was isolated using an Immunopure protein A IgG orientation kit (Pierce, Rockford, IL). Sera were loaded onto columns and the columns were washed with 4 5 ml 1 m NaCl. The IgG fraction was eluted with 0.1 m glycine, pH 2, and neutralized with 100 l of 1 1 m Tris, pH 7.5. Absorbance at 280 nm was taken, and the concentration was decided using the equation 1 OD = 0.75 mg/ml protein. Before culturing, preimmune and immune IgG fractions were dialyzed against sterile RPMI medium overnight at 4C. The IgG fractions were placed in 6C8 kDa dialysis tubing, which was placed in 500 ml of sterile RPMI medium. After overnight incubation, the medium was refreshed once, and dialysis continued for another 5 hr. Fifty nanograms of purified fusion protein was electrophoresed at 200 V in a 7.5% SDS-PAGE gel.
