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.
Np2 micromanipulator
Manufactured by Eppendorf
The NP2 micromanipulator is a precision instrument designed for fine and accurate positioning of objects or samples in a laboratory setting. It provides precise control and movement capabilities to assist with various experimental and research tasks. The core function of the NP2micromanipulator is to enable precise and delicate manipulation of small-scale objects or samples in a controlled manner.
Automatically generated - may contain errors
Lab products found in correlation
Femtojet microinjection system, by Eppendorf (2 mentions)
Hydraulic micromanipulator, by Narishige (1 mentions)
Rhodamine tubulin, by Cytoskeleton (1 mentions)
Eclipse te2000 microscope, by Nikon (1 mentions)
Tw100f 4, by World Precision Instruments (1 mentions)
Mmessage mmachine t7 in vitro transcription kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using np2 micromanipulator
1
Microinjection of Rhodamine Tubulin in Neurons
2
mRNA Microinjection in Aplysia Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mRNAs of Aplysia Src and cortactin constructs were prepared using the mMESSAGE mMACHINE T7 in vitro transcription kit (Ambion, Life Technologies, Grand Island, NY) and expressed by microinjection into Aplysia bag cell neurons typically 18–24 h after cell plating. mRNA, 3 mg/ml in 10 mM Tris, pH 8.0, 1 mM EDTA was heated at 65°C for 5 min, mixed with an equal volume of 3 mg/ml Alexa 568-G actin (Life Technologies) in G-buffer (2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, 0.5 mM dithiothreitol), and spun at 14,000 × g for 30 min at 4°C before microinjection. Microinjection was performed using the NP2 micromanipulator and FemtoJet microinjection system (Eppendorf, Hauppauge, NY). Reagent solution injection volumes were typically ∼10–15% of cell volume. After microinjection, neurons were incubated in culture medium for 48 h before imaging.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!