DMSO, 1 mM palmostatin or 100 μM Wnt-C59 were loaded into a needle pulled from a 1 mm glass capillary tube (TW100F-4, World Precision Instruments) using a P-87 Micropipette Puller (Sutter Instrument). Embryos were placed in a mesh-bottomed dish with 1/10X MMR 3% Ficoll and microinjected in both blastomeres with 1 nl of the appropriate solution using a Picospritzer III microinjection system (Parker) equipped with a MM-3 micromanipulator (Narishige). Injected embryos were transferred to a new dish and incubated at 23°C in 1/10X MMR 3% Ficoll until stages 6–9, then processed for immunostaining.
Mm 3 micromanipulator
Manufactured by Narishige
Sourced in Japan
The MM-3 micromanipulator is a high-precision positioning device designed for use in various laboratory applications. It offers precise control and positioning of samples or instruments with micrometer-level accuracy. The MM-3 is a compact and durable unit that can be integrated into a wide range of experimental setups.
Automatically generated - may contain errors
4 protocols using mm 3 micromanipulator
1
Wnt Pathway Inhibition in Xenopus Embryos
2
Orthotopic Implantation of Tumor Cells in Mouse Brain
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Tumor cells (10,000 cells/µl) were re-suspended in Matrigel (Corning, NY, USA) and TSM (1:1) and injected (1 µl) using Narishige MM-3 micromanipulator (Tokyo, Japan). The cells were systematically injected in parallel into both the brainstem and cortex of P2–4 mouse brains, two injections per region. The injections of each region were performed consecutively, alternating the order of injections between brainstem and cortex. After injections, the brain regions were gently separated and tissue pieces were incubated on top of 40-µm cell filters. Each well was filled with medium (Neurobasal, 15 mg/ml glucose, AmphoB 25 µg/ml, Pen-Strep, and B27 [without vitA]) up to the level of the filter to allow diffusion. Incubation was for 24–72 h in 5% CO2, 37° C.
3
Microinjection of 2-cell Embryos
4
Overexpression of Transcription Factors in Embryos
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Stage 1 (one-cell) embryos about 30 min post-fertilization were transferred into a mesh-bottom dish containing 1/9X MMR 3% Ficoll for microinjection. Injections were done using a Picospritzer III microinjection system (Parker, Hollis, NH) equipped with a MM-3 micromanipulator (Narishige, Amityville, NY). To mimic overexpression of each transcription factor, each embryo was injected with 750 picograms of mRNA, a dose we determined was large enough to see phenotypes, but was not associated with embryo toxicity. Injected embryos were transferred to a new dish coated with 1.5% agarose in 1/10x MMR, and incubated at 23°C in 1/9X MMR 3% Ficoll for at least 6 h. The embryos were then transferred to fresh 1/10x MMR in a new agarose-coated dish, and incubated at 23°C with buffer changes into fresh 1/10x MMR several times daily until ready for fixation or imaging.
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