For chick neurons, microinjection pipettes (TW100F-4, World Precision Instruments, Inc. Sarasota, FL) were pulled on a Brown and Flaming horizontal pipette puller. For Aplysia neurons, microinjection pipettes (1B100F-4, World Precision Instruments) were pulled on a Narishige PP830 vertical pipette puller. Pipettes were then back-loaded with 1 mg/ml rhodamine tubulin (Cytoskeleton, Inc., Denver, CO, USA) in injection buffer (100 mM PIPES pH 7.0, 1 mM MgCl2, 1 mM EGTA) as previously described84 . Before injection, tubulin was thawed, spun at 13,000 g for 30 min, kept on ice and back-loaded into pipettes pre-chilled to 4 °C. Microinjection was performed using the NP2 micromanipulator and FemtoJet microinjection system (Eppendorf North America, New York, NY), visualized with a Nikon ECLIPSE TE2000 microscope using phase contrast optics with a 40x objective. Alternately, microinjection was performed using a Narishige hydraulic micromanipulator with injection pressure supplied from a 3 ml luer-lock syringe.
Rhodamine tubulin
Manufactured by Cytoskeleton
Sourced in United States
Rhodamine tubulin is a fluorescently labeled tubulin protein used for visualizing microtubule structures in live cell imaging. It is a core cytoskeletal component essential for a variety of cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 microscope, by Nikon (1 mentions)
Hydraulic micromanipulator, by Narishige (1 mentions)
Femtojet microinjection system, by Eppendorf (1 mentions)
Tw100f 4, by World Precision Instruments (1 mentions)
Np2 micromanipulator, by Eppendorf (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Lsm 710 confocal laser scanning system, by Zeiss (1 mentions)
Observer z1, by Zeiss (1 mentions)
Halocarbon oil 700, by Merck Group (1 mentions)
Bleomycin, by Merck Group (1 mentions)
4 protocols using rhodamine tubulin
1
Microinjection of Rhodamine Tubulin in Neurons
2
Visualizing Motor-Driven MT Bundling
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Fluorescent MTs were polymerized by incubating 25 mM tubulin (Cytoskeleton Inc.)+1 mM rhodamine tubulin (Cytoskeleton Inc.) with 1 mMGTP and 10 mM taxol in BRB80 (80 mM K2PIPES,1 mM EGTA, 1 mM MgCl2)+10% glycerol at 37 °C for 35 min. Bundling assays were performed in buffer T (20 mM Tris, pH 8.0, 150 mM KCl, 2 mM MgCl2, 1 mM DTT, protease inhibitors) with 2.5 μM MTs, 0.2 μM motor proteins, 10 μM taxol and 1 mM ATP (final concentrations). The mixture was rocked at 22 °C for 20 min and transferred into a flow chamber with a DEAE–Dextran-coated coverslip such that the unstuck proteins were washed out. Bundling of fluorescent MTs was observed with an inverted widefield fluorescent microscope. Each experiment was repeated 3 times, and each time ⩾10 slides were checked to ensure consistent results.
3
Kinetochore Assembly and Checkpoint Assay
4
Embryo Injection for DNA Damage Study
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Embryo injection to cause DNA damage was performed as previously described (Takada et al., 2003 (link); Takada and Cha, 2011 (link)). Briefly, 0- to 2-h-old embryos were hand dechorionated, slightly dehydrated on Dri-rite (W. A. Hammond, Xenia, OH) for 2–4 min, mounted on a glue-coated coverglass, and covered with Halocarbon oil 700 (Sigma-Aldrich). The embryos on the coverglass were placed on Zeiss Observer Z1 inverted fluorescence microscope. For functional rescue experiments, 25 μg/ml bleomycin (Sigma-Aldrich), which consistently causes nuclear dropping, severe centrosome inactivation, and mitotic delay in wild-type embryos, was coinjected with 5 mg/ml rhodamine-tubulin (Cytoskeleton, Denver, CO) in BRB80 (80 mM Na–1,4-piperazinediethanesulfonic acid, pH 6.9, 1 mM MgCl2, 1 mM ethylene glycol tetraacetic acid, and 1 mM GTP). Injection was manually performed with a glass needle connected to a 10-ml syringe with a narrow tube and mounted on a micromanipulator. Rhodamine signal representing microtubule structures and EGFP signal representing localization of EGFP-tagged protein were captured simultaneously starting immediately after the injection with a Zeiss LSM710 laser-scanning confocal system and α Plan-Apochromat 100×/1.46 Oil DIC objective lens with 10-s intervals. Image acquisitions and analyses were performed with ZEN and ImageJ.
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