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Tsien, University of California San Diego, La Jolla, CA). Plasmid DNA (??2?��g/��l) was dissolved in a solution of 10?mM Hepes (pH 7.4) and 10?mM KCl with 0.5% Alexa 488 or 568 dextran (Molecular Probes) to visualize the injectate. For injections, embryos were anesthetized in a solution of 8% ethanol in sterile artificial pond water and placed ventral side up long a trough cut into a Sylgard coated dish (184 silicone; Dow Corning Co.). The ventral surface of individual ganglia Thiazovivin in vivo was exposed by making a small incision in the body wall (Icotinib mouse siRNA Cocktail kit (Ambion) as previously described (Baker & Macagno, 2000?and?Baker et al., 2008). For HmLAR1, dsRNAs encompassed nucleotides 1�C950 of the DNA template. For HmLAR2, dsRNAs encompassed nucleotides 1�C1650 of the DNA template as described previously ( Baker et al., 2008). Injections of identified central neurons were performed as described above for transgene YES1 expression, except that the solution was injected into the cytoplasm. Leech embryos used in these experiments were obtained from a mixed Hirudo medicinalis and Hirudo verbana colony maintained in our laboratory. Prior to use, they were removed from their cocoons and kept in artificial spring water (0.5?g/l Instant Ocean, Aquarium Systems) at 22�� C, and staged according to the criteria of Fernadez and Stent (1982). At this temperature, day 0 (E0) is defined as the day of cocoon deposition and day 30 (E30) as the day of emergence of the juvenile animal from the cocoon. All expression and knockdown experiments were performed on embryos beginning at day 12�C13. Anti-GFP an HA immunocytochemistry was performed on whole mounted embryos using a directly conjugated Alexa 488, rabbit anti-GFP antibody (Molecular Probes), a monoclonal anti-HA antibody (A2095; Sigma) and an anti-mouse antibody conjugated to Alexa 568 or 633 (Molecular Probes), using established methods (Baker et al., 2000). The effectiveness of all RNAi knockdown injections was confirmed through in situ hybridization staining ( Nardelli-Haefliger and Shankland, 1993).