Zebrafish used in this study were housed in the animal facility at Brandon University, maintained by Dr Christophe LeMoine. The wild-type adult zebrafish (Danio rerio) were bred in a plastic tank maintained with Brandon dechlorinated tap water in a 10 h:14 h light dark cycle at 28°C. The fish were fed once daily on Adult Zebrafish Complete Diet (Zeigler, Gardners, PA). The fertilized zebrafish eggs were harvested in hundreds and eggs were raised (one-cell stage) in a glass-plated petri dish filled with E3 embryo medium (in mM: 5 NaCl, 0.17 KCl, 0.33 CaCl, 0.33 MgSO4 and 0.00001% Methylene Blue) and kept at 28°C. With the aid of a microinjector (IM 300, Narishige, Long Island, USA) and a pulled 1.0-mm borosilicate glass micropipette (Stutton Instrument, Novato, USA), 1 nl volume of either PGE2 (4 μM) or vehicle (0.13% BSA) along with 1μM of red fluorescent dye (Dextran, Texas Red™, 3000 MW, Lysine Fixable, Thermo Fisher Scientific) were injected into eggs. Following the microinjection, all groups including PGE2-injected, vehicle-injected and the non-injected control were maintained under the same conditions in E3 growth media and incubated at 28°C (LeMoine and Walsh, 2013 (link)). Phenotypic changes during embryonic growth of zebrafish were monitored and recorded until 96 hpf with a stereoscopic and a fluorescence microscope.
Im 300
Manufactured by Narishige
Sourced in Japan, United States
The IM-300 is a microinjector manufactured by Narishige. It is designed to precisely control the injection of small volumes of fluid into biological samples, such as cells or tissues. The IM-300 allows for accurate and reproducible injection of fluids through the use of a microprocessor-controlled system.
Automatically generated - may contain errors
Lab products found in correlation
Angle two software, by Leica (2 mentions)
Ms 222, by Merck Group (2 mentions)
Cm dil, by Thermo Fisher Scientific (2 mentions)
Lysine fixable, by Thermo Fisher Scientific (1 mentions)
Ahr2 mo, by Gene Tools (1 mentions)
Alexa 594 conjugated dextran, by Thermo Fisher Scientific (1 mentions)
Cellmask orange, by Thermo Fisher Scientific (1 mentions)
Axiovert 100, by Zeiss (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Stereo discovery v20, by Zeiss (1 mentions)
Truecut cas9 protein v2, by Thermo Fisher Scientific (1 mentions)
Fluorescence stereomicroscope, by Olympus (1 mentions)
G mops plus, by Vitrolife (1 mentions)
Fluorinert fc 770, by Merck Group (1 mentions)
Alexa fluor 647 labeled dextran, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Dnase treatment, by Thermo Fisher Scientific (1 mentions)
Morpholino, by Gene Tools (1 mentions)
P7629, by Merck Group (1 mentions)
29 protocols using im 300
1
Zebrafish Embryonic Development Assay
2
Morpholino Antisense Oligonucleotides in Zebrafish
3
Retrograde Labeling of Spinal Neurons
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After the embryos were decapitated and eviscerated, the target muscles and nerves were exposed in cold PBS, and Alexa594-conjugated Dextran (Cat# D22913, Thermo Fisher Scientific, USA) was injected using a picosplitzer (IM300, Narishige, Japan). The injected embryos were kept in oxygen-bubbled DMEM (Wako Pure Chemical, Japan) for up to 5 h at 30°C. After incubation, the embryos were fixed in 4% PA in 0.1 M PB for 5 h and then immersed in 20% sucrose in PBS overnight. The appropriate segments of the spinal cord were excised from the embryo and frozen-embedded in a mixture of 20% sucrose in PBS and Tissue-Tek embedding media (1:2 ratio; Sakura, Japan). Ten to fifteen muscles were injected with the tracer, and one-to six spinal cords with successful labeling were obtained for each muscle type.
4
Microinjection of Oocytes and Fluorescent Imaging
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For microinjection dechorionated oocytes were mounted in glass wedges and injected with mRNA (1-2 µg/µl pipette concentration/~1–2% injection volume) using a high pressure system (Narishige IM300)64 (link). mRNA-injected oocytes were left for 2–5 h or overnight before fertilization and imaging of fluorescent fusion protein constructs. The lipophilic dye Cell Mask Orange (Molecular Probes) was prepared at a concentration of 10 mg/ml in DMSO and diluted in sea water at 20 µg/ml then mixed 1:1 with the embryos just prior to imaging. Epifluorescence imaging was performed with an Olympus IX70, Zeiss Axiovert 100, or Axiovert 200 equipped with cooled CCD cameras and controlled with MetaMorph software package. Confocal microscopy was performed using a Leica SP5 or SP8 fitted with 40×/1.3NA oil objective lens and 40×/1.1NA water objective lens, respectively. All live imaging experiments were performed at 18–19 °C. For fast imaging of cortical preparations a rectangular image section of the imaging array was selected to increase the temporal resolution to 0.8 images/sec. Image analysis was performed using Image J, ICY and MetaMorph software packages. Calcium-free sea water: 450 mM NaCl, 9 mM KCl, 33 mM Na2SO4, 2.15 mM NaHCO3, 10 mM Tris pH 8, and 2.5 mM EGTA.
5
Sciatic Nerve Tumor Implantation and Treatment
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Human SC-WT or SC-NF2-null cells were injected directly into the sciatic nerve of isoflurane-anesthetized mice, as described.36 (link) Specifically, cells were implanted approximately 4 mm distal to the sciatic notch at a point midway between the sciatic notch and the trifurcation of the sciatic nerve into the common peroneal, tibial, and sural branches. Cells were first trypsinized and rinsed, and 30,000 cells in 2 μL of culture medium were injected into the distal sciatic nerve of athymic nude mice (nu/nu, 4–6 weeks old males and females; National Cancer Institute [NCI]) using a glass micropipette and a gas-powered microinjector (IM-300; Narishige, Tokyo, Japan). Tumor growth was monitored by in vivo BLI at weekly intervals, as described.45 (link) Briefly, mice were injected intraperitoneally with the Fluc substrate D-luciferin and, 5 min later, signal was acquired with a high-efficiency IVIS Spectrum (Caliper Life Sciences, Hopkinton, MA) under an XGI-8 gas anesthesia system (Caliper Life Sciences). After 4 weeks, tumors that grew progressively were injected with AAV1-CBA-FLAG-merlin (4 × 108 gc) in 2 μL of PBS. Volumetric changes in tumors were tracked by in vivo BLI out to 10 weeks post vector injection.
6
Establishing Ectopic Retinoic Acid Sources
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MOs were injected at the one-cell stage and cell transplantations were performed as previously described (White et al., 2007 (link); Cai et al., 2012 (link)).
For the generation of ectopic retinoic acid (RA) sources, mineral oil was infused with all-trans RA to saturation. Embryos were dechorionated and temporarily mounted in 1% low-melt soft agar in EM over coverslips. Drops of the RA saturated oil or oil alone were then injected in 6–12 embryos using a mouth pipette and a capillary needle. The embryos were then released and left to heal for two hours when they were mounted for FLIM imaging. This experiment was repeated 3 (three) times.
mRNA was injected into one-cell embryos with glass micropipettes and a Narishige IM 300 microinjector with 50 pg of GFP-CAAX, 50 pg of Crabp2a-Myc or 100 pg of Cyp26a1-Myc. Expression verification was performed by microscopic observation for GFP or by Western blot with anti-Myc antibody (clone 9E10) for Crabp2a-Myc and Cyp26a1-Myc.
For the generation of ectopic retinoic acid (RA) sources, mineral oil was infused with all-trans RA to saturation. Embryos were dechorionated and temporarily mounted in 1% low-melt soft agar in EM over coverslips. Drops of the RA saturated oil or oil alone were then injected in 6–12 embryos using a mouth pipette and a capillary needle. The embryos were then released and left to heal for two hours when they were mounted for FLIM imaging. This experiment was repeated 3 (three) times.
mRNA was injected into one-cell embryos with glass micropipettes and a Narishige IM 300 microinjector with 50 pg of GFP-CAAX, 50 pg of Crabp2a-Myc or 100 pg of Cyp26a1-Myc. Expression verification was performed by microscopic observation for GFP or by Western blot with anti-Myc antibody (clone 9E10) for Crabp2a-Myc and Cyp26a1-Myc.
7
Chemogenetic Modulation of S1 Cortex
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Each isoflurane-anesthetized TRAP mouse received an i.p. injection of CNO (4 mg/kg body weight; Enzo) before behavior tests were conducted (Figures 3 , 4 , 5 , 6 , and 7 ) to activate or inhibit DREADD-expressing neurons in the S1 cortex. In the experiments shown in Figures 6 and 7 , injection of CNO was performed before or after intradermal injection of CGN into the right hind paw (1%, 20 μl; Sigma-Aldrich).28 (link),29 (link) Moreover, Fo-TRAP-Gq mice received an i.p. injection of CNO after administration of saline (1 μl) or TTX (5 μM, 1 μl) into the Pf (−2.1 mm posterior and −0.77 mm lateral to the bregma; −3.3 mm from the brain surface) using a microinjector (IM 300, Narishige) in the experiment shown in Figure 7 .
8
Tracking A549 Cell Migration in Zebrafish
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After anesthetization, 100 to 200 CM-Dil (a lipophilic fluorescent tracking dye) labelled A549 cells were grafted into yolk sac of each wild-type zebrafish embryos at 48 hpf with a microinjector IM-300 (Narishige, Tokyo, Japan). In vivo imaging was performed at 5dpf. After injection, embryos were incubated for 1 h at 28 °C, then at 35 °C.
9
Stereotaxic Viral Injections in Mice
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Stereotaxic injections into the striatum and caudal intralaminar thalamic nuclei were performed with the guidance of Angle Two software (Leica Biosystems, Buffalo Grove, IL). Mice were anesthetized with isoflurane. The position of bregma and lambda were provided to the software, and then a glass pipette was slowly inserted through a burr hole to the coordinates calculated by the software. The AAV virus was slowly injected by using a microinjector (NARISHIGE IM300, Japan) or syringe into the CLN; coordinates relative to bregma (in mm) were: lateral, 0.78; posterior, 1.34; depth, 3.00/2.80. Mice were analyzed 4 weeks after virus injection. All mice included in the study had postmortem verification of the viral injection into the CLN.
10
mRNA and DNA Microinjection in Xenopus
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A total volume of 10 nl of mRNA and/or plasmid DNA was injected into X. laevis embryos at the 1-cell, 2-cell or 4-cell stage using a microinjector (Narishige IM-300). Microinjection needles were produced from borosilicate glass capillaries (Harvard Apparatus, GC120-15) using a micropipette puller (Sutter p97).
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