To assess whether GO can cause fatal damage by spreading through the bloodstream of the larvae, various concentrations of GO were introduced into the systemic circulation by microinjection in the duct of Cuvier (DC) according to the following protocol: larvae were anesthetised in zebrafish water containing 160 μg mL−1 MS-222 (Sigma-Aldrich) and placed on an agarose plate to be individually microinjected with 2 nL of selected GO concentrations using a glass microneedle from the Narishige MN-151 micromanipulator and a FemtoJet 4x microinjector (Eppendorf). Only the two highest GO concentrations of 0.25 and 0.1 mg mL−1 were tested by this route. After microinjection, larvae were maintained at 28 °C, and their mortality was recorded. The resulting data were used to calculate the survival rate. Each assay was performed twice, on a total of 30 embryos per condition, and with the reference of a control based on distilled water.
Mn 151
Manufactured by Narishige
Sourced in Japan
The MN-151 is a microtranslator produced by Narishige, a leading manufacturer of high-quality laboratory equipment. The core function of the MN-151 is to provide precise and controlled translation of micromanipulators or similar devices used in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ribomax large scale rna production system t7, by Promega (3 mentions)
Ms 222, by Merck Group (2 mentions)
Pmd18 t plasmid, by Takara Bio (2 mentions)
Halocarbon oil 700, by Merck Group (2 mentions)
Femtojet 4x microinjector, by Eppendorf (1 mentions)
Dna gel purification kit, by Omega Bio-Tek (1 mentions)
Antibiotic antimycotic mixed stock solution, by Nacalai Tesque (1 mentions)
Gtbp01300, by Merck Group (1 mentions)
Dna purification kit, by Omega Bio-Tek (1 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Santa Cruz Biotechnology (1 mentions)
Csu 10, by Zeiss (1 mentions)
Halocarbon oil 27, by Merck Group (1 mentions)
α amanitin, by Santa Cruz Biotechnology (1 mentions)
T7 ribomax express rnai system kit, by Promega (1 mentions)
P 1000 flaming brown micropipette puller, by Sutter Instruments (1 mentions)
Borosilicate glass capillaries, by Harvard Apparatus (1 mentions)
11 protocols using mn 151
1
Assessing Lethal Effects of Graphene Oxide in Zebrafish Larvae
2
RNAi Silencing of NlMet and NlKr-h1 in Nymphs
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For the RNAi experiment, the double-stranded RNA (dsRNA) was synthesized using the synthesis kit RiboMAX™ Large-Scale RNA Production System-T7 (Promega, Beijing). The templates for the synthesis of the dsRNA were amplified by PCR using the PMD18-T plasmid (Takara, Dalian, China) with a DNA fragment of the gene of interest inserted, and then the PCR products were purified with a DNA gel purification kit (Omega bio-tek, USA). The dsRNAs were synthesized as described in the Promega technical bulletin TB166. The primers used for the dsRNA synthesis of NlMet, NlKr-h1 and the control gene encoding Green Fluorescent Protein (GFP) are listed in Table 2 .
The 5th instar nymphs were anaesthetized with CO2 before the intra-thoracic dsRNA injection. The injection was conducted using a Nikon microscope and a Narishige injection system (MN-151; Narishige). In total, 0.1 μg of dsRNA was injected into each insect. The nymphs were allowed to recover for 1–2 h after the injection and then were reared on rice seedlings. The mortality, wing-morph and sex were recorded after injection, and then the females were used for ovary dissections or were prepared for total RNA extraction. Three independent biological replicates were used for the nymphs that were injected with dsNlMet, dsNlKr-h1, both dsN1Met and dsN1Kr-h1, or only dsGFP.
The 5th instar nymphs were anaesthetized with CO2 before the intra-thoracic dsRNA injection. The injection was conducted using a Nikon microscope and a Narishige injection system (MN-151; Narishige). In total, 0.1 μg of dsRNA was injected into each insect. The nymphs were allowed to recover for 1–2 h after the injection and then were reared on rice seedlings. The mortality, wing-morph and sex were recorded after injection, and then the females were used for ovary dissections or were prepared for total RNA extraction. Three independent biological replicates were used for the nymphs that were injected with dsNlMet, dsNlKr-h1, both dsN1Met and dsN1Kr-h1, or only dsGFP.
3
RNAi Knockdown of Insect Genes
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dsRNAs of brown planthopper Kr-h1 and E93 were synthesized using the RiboMAX™ Large Scale RNA Production System-T7 (Promega, Madison, WI, USA). The synthesis procedure was the same as Technical Bulletin TB166 (Promega), except that different templates and primers were used for synthesizing different dsRNA (with 27 bases at 5′, Table 1 ).
Nymphs of the appropriate developmental stage were anaesthetized by CO2. A Nikon microscope and Narishige injection system (MN-151, Narishige Scientific Instrument Lab, Tokyo, Japan) were used for injection; 3rd, 4th and 5th instar nymphs were anaesthesized by CO2 and 0.1 μg (0.2 μL) dsRNA was injected into the abdomen of each nymph. The nymphs were recovered for 2 h after injection and cultured on rice seedlings. The same amount of dsRNA was used for the dual knock-down experiment (0.1 μg for each dsRNA) [25 (link)].
Nymphs of the appropriate developmental stage were anaesthetized by CO2. A Nikon microscope and Narishige injection system (MN-151, Narishige Scientific Instrument Lab, Tokyo, Japan) were used for injection; 3rd, 4th and 5th instar nymphs were anaesthesized by CO2 and 0.1 μg (0.2 μL) dsRNA was injected into the abdomen of each nymph. The nymphs were recovered for 2 h after injection and cultured on rice seedlings. The same amount of dsRNA was used for the dual knock-down experiment (0.1 μg for each dsRNA) [25 (link)].
4
Labeling Shark Embryos with CM-DiI
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For the injection of shark embryos at stage 25, eggs were removed from the seawater tank and briefly incubated on ice. A small window was opened on the surface of the egg case just above the embryo. Embryos were then anesthetized with 20 μl of a mixed solution of 1% ethyl 3-aminobenzoate methanesulfonate (MS-222) (E10521; Sigma) and 2% sodium carbonate (1:1 volume:volume). CM-DiI (C7001; Thermo Fisher Scientific, USA) was prepared as previously described [29 (link)] and microinjected into HCs by using a microinjector (MN-151; Narishige, Japan). After injection, 200 μl of 0.2% antibiotic-antimycotic mixed stock solution (09366–44; Nacalai Tesque, Inc., Japan) in PBS was added to the surface of the embryo. The eggshell was sealed with a polycarbonate filter (GTBP01300; Merck Millipore, USA) using cyanoacrylate adhesive (Aron Alpha, also known as ‘Krazy Glue’; Toagosei, Japan), to prevent air bubbles from entering the eggshell and to prevent the contents of the egg from protruding through the opening. The injected embryos were left to develop for 6–7 weeks at 16 °C in seawater with 0.2% antibiotic-antimycotic mixed stock solution without aeration. The incubation seawater was replaced multiple times weekly.
5
Dsrna Injection in N. lugens
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Nymphs were anesthetized with carbon dioxide, and a Nikon microscope and Narishige injection system (MN-151, Narishige Scientific Instrument Lab, Tokyo, Japan) were used for injection. Then 0.1µg (0.2µl) of dsRNA was injected into anesthetized N. lugens. After 2 h of recovery, the nymphs were transferred and cultured with rice seedlings (Liu et al. 2010 (link), Li et al. 2011 (link)).
6
Dsrna-mediated Gene Silencing in Insects
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Double stranded RNA was synthesized using the synthesis kit RiboMAX™ Large Scale RNA Production System-T7 (Promega, Beijing). The template of dsRNA synthesis was amplified by PCR using the PMD18-T plasmid (Takara, Dalian) inserted with a DNA fragment of the gene of interest, and then the PCR fragment was purified with a DNA purification kit (Omega bio-tek, USA). dsRNA synthesis was carried out as described in the Promega technical bulletin TB166 and in our previous paper 52 (link), 53 . Primers used for dsRNA synthesis, including primers for the control gene Green Fluorescent Protein (GFP), are listed in Table 1 .
dsRNA was injected into the thorax of CO2-anesthesized 4th and 5th instar nymphs using a Nikon microscope and Narishige injection system (MN-151, Narishige) as previously described 54 (link). 0.1 μg dsRNA was injected for each insect. The concentration and volume of the dsRNA injections were chosen based on previous studies 53 , 54 (link). Nymphs were reared on rice seedlings after injection and cultured under conditions described above. The percentage of individuals developing with long and short wings was then compared between treatment and control populations using Chi Square tests in SPSS 20.0.
dsRNA was injected into the thorax of CO2-anesthesized 4th and 5th instar nymphs using a Nikon microscope and Narishige injection system (MN-151, Narishige) as previously described 54 (link). 0.1 μg dsRNA was injected for each insect. The concentration and volume of the dsRNA injections were chosen based on previous studies 53 , 54 (link). Nymphs were reared on rice seedlings after injection and cultured under conditions described above. The percentage of individuals developing with long and short wings was then compared between treatment and control populations using Chi Square tests in SPSS 20.0.
7
Zebrafish trim45 mRNA Overexpression
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Open reading frame (ORF) sequences of zebrafish trim45 were synthesized using following primers: trim45-ORF-forward primer – 5′-GCGGATCCATGTCACTTTGTAAAGAGAAGGG-3′ and trim45-ORF-reverse primer – 5′-ATATGATATCGAGCTCCACAGTGCGGAGGT-3′. The synthesized DNA fragment was cloned into pGEM-T easy vector and digested by BamHI and EcoRV restriction enzymes. The isolated cDNAs containing trim45 mRNA were ligated with pcGlobin2 vector (Ro et al. 2004 (link)) which was linearized by BamHI and EcoRV. The trim45 ORF + pcGLobin2 construct was linearized by XbaI enzyme and subjected to in vitro transcription using the mMessage mMachine T7 kit (Ambion). Approximately, 50–100 ng of trim45 mRNA (200 ng–400 ng/μl) was injected into embryos at one cell stage using a micromanipulator (Narishige MN-151).
8
Live Imaging of Embryos with Pharmacological Manipulations
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Live imaging of embryos was conducted using a Yokogawa CSU10 spinning disc confocal microscope (Zeiss Observer.A1). Embryos were dechorionated in 4% bleach, washed with water, and mounted in halocarbon 27 Oil (Sigma). All live images were acquired in one minute intervals using Planapochrom 63X 1.4NA Oil objective. For pharmacological manipulations, embryos were lined up and glued to a coverslip, and desiccated for 12 to 13 minutes in a closed chamber containing drierite. Following desiccation, embryos were covered in halocarbon 700 oil (Sigma). Embryos were subsequently injected using a micromanipulator (Narishige, MN-151) with either water, 10,000 μg/mL puromycin (Santa Cruz Biotechnology), 1,000 μg/mL microbially sourced cycloheximide (Sigma), or 100 μg/mL α-amanitin (Santa Cruz Biotechnology) and imaged.
9
RNAi Knockdown Assay in Brown Planthopper
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A PCR product with a size about 500 bp was used to synthesize double-stranded RNA (dsRNA). The primers were shown in Table S3 . Region of dsRNA and qRT-PCR primers was shown in Figure S2 . GFP dsRNA was used as a control. dsRNA was synthesized according to the instructions of the T7 RiboMAX Express RNAi System kit (Promega, Madison, WI, USA), and frozen at −20 °C for later use.
The nymphs were injected with 0.2 μg dsRNA of the corresponding gene using the Narishige Injection System (MN-151, Narishige, Japan). The control group was injected with the same amount of GFP dsRNA. After injection, the brown planthoppers were allowed to recover for 2 h and then cultured on rice seedlings. The brown planthoppers were cultured in an incubator in groups of 10. After RNA extraction, cDNA was synthesized and qRT-PCR was used to measure the expression level of genes. The results were analyzed using the 2−ΔΔCt relative expression method [29 (link)]. In addition, the survival rate and molting rate were calculated.
The nymphs were injected with 0.2 μg dsRNA of the corresponding gene using the Narishige Injection System (MN-151, Narishige, Japan). The control group was injected with the same amount of GFP dsRNA. After injection, the brown planthoppers were allowed to recover for 2 h and then cultured on rice seedlings. The brown planthoppers were cultured in an incubator in groups of 10. After RNA extraction, cDNA was synthesized and qRT-PCR was used to measure the expression level of genes. The results were analyzed using the 2−ΔΔCt relative expression method [29 (link)]. In addition, the survival rate and molting rate were calculated.
10
Microinjection of C. capitata Embryos
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For microinjection of homozygous C. capitata TREhs43hid Ala5 _F1m2 embryos eggs were collected over a 45-90 min period. Eggs were prepared for injection as previously described [41] using chemical dechorionization (sodium hypochlorite, 3 min). In brief, embryos were fixed using double-sided sticky tape onto a microscope slide (Scotch 3M Double Sided Tape 665), and covered with halocarbon oil 700 (Sigma-Aldrich, Munich, Germany). Injections were performed using borosilicate needles (GB100F-10 with filaments; Science Products, Hofheim, Germany), drawn out on a Sutter P-2000 laser-based micropipette puller. The injection station consisted of a manual micromanipulator (MN-151, Narishige), an Eppendorf femtoJet 4i microinjector, and an Olympus SZX2-TTR microscope (SDF PLAPO 1xPF objective). The microscope slide with the injected embryos was placed in a Petri dish containing moist tissue paper in an oxygen chamber (max. 2 psi) and stored at 25°C, 60% RH for 72 hr to allow larval hatching. Hatched first instar larvae were transferred from the oil to larval food.
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