Multisample embedding and necessary microscope modifications. Agarose, low gelling temperature 39346811 sigmaaldrich. Cloning from low melt agarose dpni cleavagemediated site directed mutagenesis c. Zebrafish embryos are ideal for imaging posterior body elongation as their. Dissolve the low melting point agarose to a final concentration of 1. How to mount zebrafish in agar zebrafish in the classroom. Agarose is highly biocompatible and possesses variable mechanical and diffusion properties. Anesthetize the embryoslarva for mounting in agar following the how to use anesthetics on zebrafish protocol.
Freeze substitution is a previously described technique for the gentle fixation and dehydration of tissue. Choose certified low melt agarose for applications that require a high resolving capacity for dna fragments. Throughout this chapter, we defer to the zebrafish book westerfield 2000 on. It is usually used for the isolation of separated dna fragments. Confocal imaging of live larval zebrafish for assessing peripheral. Low melting or low gelling temperature agarose is produced by hydroxyethylation of agarose. Melt 1% low melt agarose in a microwave or water bath.
Feb 14, 2020 then the animal was embedded in a cylinder of agarose 2% low melt agarose, 2% ethanol, in artificial seawater using a procedure similar to that used for zebrafish. A toolbox to study epidermal cell types in zebrafish. Agarose lowmelting, 1kb dnarnagenetic analysis grade. Add hepes and ms222 to final concentrations of 10 m m and 0. Usa juvenile 25 dpf in a block of 5% agarose low melting point. Twophotonbased photoactivation in live zebrafish embryos. Prepare 2% low melting point agarose ultra pure lmp agarose, invitrogen, carlsbad, ca, cat. Dechorionated embryos in embryo medium were anesthetized with tricaine sigma as per the zebrafish book. Determine the amount of agarose solution needed to cast your gel. Lowcost silicone imaging casts for zebrafish embryos and. Imaging the early zebrafish embryo centrosomes following. This solution was brought to a gentle boil in a glass beaker to solubilize agarose. Jun 15, 2011 fish were then released and allowed to recover for at least 1 h. Jan 22, 2014 due to their size and optical clarity, zebrafish embryos have long been appreciated for their usefulness in timelapse confocal microscopy.
To mount embryos, pick up one donor and one host in the same pipette, drop them briefly in the agarose to wash off the e2, then place them back in the pipette and put them in a drop of agarose on an inverted 35mm petri dish lid. Agarose covering the tail was carefully removed to allow the tail to move freely. Correlative light and electron microscopy of rare cell. It is easy to change the surrounding medium with it without displacing the embryos particularly useful if you are planning. Ultrapure low melting point agarose is ideal for resolving dna and rna fragments and for the recovery of nucleic acid fragments after electrophoresis, as it melts at 65. A guide for the laboratory use of zebrafish danio rerio. This served to protect the body, but not the head, from. The agarose should be dissolved in distilled water by heating it in pulses with a conventional microwave. Quickly suck up the embryo in a small drop of agar and transfer it o. Automated highthroughput heartbeat quantification in medaka and.
Jul 27, 2017 to perform in vivo imaging of neutrophil recruitment to the site of injection, anesthetized zebrafish larvae were oriented and immobilized in 1% low melting point agarose and covered with 0. Zebrafish mating and embryo collection zebrafish mating is coordinated with the daily lightdark cycle, generally 14 hours light and 10 hours dark, and mating will typically occur in the morning when lights are turned on. Mounting zebrafish larvae for microscopy chop research institute. Low melting point lmp agarose gel preparation protocol. Agarose should be warm enough to remain liquid, but not hot enough to harm live embryos. Sectioning lowmeltingpoint agarose lmaembedded tissue. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274. I usually add approximately ul of low melt agarose in each well. A schematic of single zebrafish embryo embedding in a fluorinated polypropylene fep tube using 2% low melting point lm agarose as a plug, 0.
Hold the gauze by the other end with forceps and plunge the embryos into the center of the isopentane for 1 min. They can be put on a drop of methyl cellulose, a good technique for an upright microscope, or they can be mounted in low melting point agarose or under thin. Short wavelength uv light will increase the chance of damaging the dna. Introduction of the interactive atlas of zebrafish. Lens transplantation in zebrafish and its application in. Promotes quick and thorough digestion of gel slices during extraction procedures. To melt the agarose in solutions of less than 2%, heat the slurry in microwave oven on high power setting until it starts to boil.
For more precise orientation of zebrafish embryos and to keep them from floating. Agarose can be used as a gelling agent, to separate nucleic acids electrophoretically. Remove the flask from the microwave oven, and gently swi rl to mix the agarose solution. Analyzing axon guidance in the zebrafish retinotectal system. Pdf cloning and characterization of a highly repetitive. For ingel applications such as digestion, ligation, pcr, transformation, and sequencing. The undigested or s1digested dna was analyzed on 1% pfgegrade low melting temperature agarose biorad, hercules, ca, and electrophoresis was performed in 0. Cells free fulltext defective excitatoryinhibitory. Quickly suck up the embryo in a small drop of agar and transfer it onto the.
This low melt agarose is recommended for preparative electrophoresis and any application requiring recovery of dna or rna. Remove the flask from the microwave oven, and gently swi rl to mix the agarose. Confocal imaging of live larval zebrafish for assessing. Coat 60 mm petri dishes with a thin layer of 1% agarose dissolved in e2 medium. Molecules free fulltext evaluation of the percutaneous. For example, morphogenesis of the zebrafish larva is investigated by embedding the fish in low melting agarose 9,10. Zebrafish danio rerio is a popular and attractive vertebrate animal model for developmental and human disease studies. A versatile mounting method for long term imaging of zebrafish.
Gentle fixation by freeze substitution gives excellent. The solution remains fluid at 37c and will set rapidly at temperatures below 25c. Allow the solution to boil for 1 min or until the solution is clear and all particles are dissolved. However, be careful of the temperature when mounting live embryos as agarose that is too hot can kill the embryos. Methodology article open access small molecule screening.
Cerebellardependent learning in larval zebrafish journal. Multisample spim image acquisition, processing and analysis. Allinone 3d printed microscopy chamber for multidimensional. Mounting the sample in low concentrations of agarose or methylcellulose in refractiveindexmatched fluorinated ethylene propylene fep tubes ensures both immobility and growth of the embryo as well as optimal image quality from all sides. Materials and methods zebrafish lines zebrafish danio rerio adults and embryos were kept at 28. Cut out the 7kb band and place in a sterile microcentrifuge tube. I originally employed it solely because it was reputed to result in minimal loss of antigenicity and had been used by other groups with similar research interests shiurba et al, 1991. Sigma aldrich and low melting point agarose agarose l. Analyzing axon guidance in the zebrafish retinotectal. The zebrafish larva is embedded in low melt agarose 1 % with the tail free to. When visualizing dna fragments to be used for ligation, use only longwavelength uv light. The settings were 6 vcm at a pulse switch time ramped from 50 to 90 s in a contourclamped. Embryos can also be dechorionated and embedded in agarose e.
Timelapse live imaging of clonally related neural progenitor. Once set, the agarose surrounding the head was removed with a scalpel to expose the head while leaving the body embedded. Zebrafish book and the zfin protocol wiki see table 1 and westerfield, 2000. Shop a large selection of electrophoresis gel casting reagents products and learn more about agarose low melting, 1kb dnarnagenetic analysis grade, fisher bioreagents. How best to immobilise zebrafish larvae for imaging. Real time observation of neutrophil white blood cell. The characteristics that make zebrafish popular include their small size, low cost, high fecundity, optical transparency in embryo and larvae stage, rapid development and generation time, and genetic and organ similarity with humans 14,15. I had good results with low melting point agarose, as suggested above.
Care was taken not to allow the holding pipette to rupture the yolk ball. Add room temperature buffer tae or tbe into a flask that can hold 24 times the volume of your agarose solution. The fep tube with the sample was then plugged with 3% low melting agarose. As hot agarose will damage the embryos, ensure that the agarose cools slightly before covering embryos.
Applied to both zebrafish and medaka we quantified native heart rate. Jun 01, 2011 the advantages of using zebrafish, which are an excellent model for research and education, include low cost, low maintenance, rapid development, ease of genetic manipulation, genomic similarities with humans, ease of external fertilization, and the possibility to produce eggs and embryos continuously with only a few fish. Small molecule screening platform for assessment of. In rather low concentrations 0,5% to 1,0% it is translucent, cheap, chemically inert and has a refractive index close to water. Allows for digestions and ligations in melted gel and easy recovery from gel slices. Our novel assay permits largescale applications ranging from phenotypic. After preparation of agarose, allow to cool to an appropriate temperature before mounting the embryos. Animal husbandry and rapid dissection of the adult zebrafish heart adult zebrafish of 46 months were anesthetized and immediately transferred to a petri dish under a microscope for dissection. Low melt agarose is a good choice for long term imaging as it keeps the embryo firmly in place. Sustained rhythmic brain activity underlies visual motion. B samples were embedded in 4% low melting point lmp agarose within a plastic mold.
Multisample spim image acquisition, processing and. Introduction of the interactive atlas of zebrafish vascular. These findings suggest that zebrafish are capable of absorbing drug. L low melting point agarose to the mattek plate coverslip. Article developing a novel embryolarval zebrafish xenograft assay t.
Dna fragments of the equal size will take longer time to move through a low melting agarose gel compared to a standard agarose gel. Adult zebrafish of 46 months were anesthetized and immediately transferred to a petri dish under a microscope for dissection. Gels, traditionally made with low melting agarose, are the most common way to mount samples for light sheet imaging. Cells of interest in red primordial germ cells pgcs in this example, marked with asterisk. Lens transplantation in zebrafish and its application in the. The solid agar should be microwave until liquidize and the temperature of the solution should be warm enough that it can be touched, not overly hot. Aliquot the agarose solution in 2 ml microcentrifuge tubes. Using two forceps dumont forcep no 5, sigma, the zebrafish was rapidly decapitated.
Millipore low melt temperature agarose gel electrophoresis low melt temperature agarose gel electrophoresis, supplied by millipore, used in various techniques. Boil until agarose is dissolved completely and add 1 ml into 42. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274 university of oregon, eugene, or 97403 usa. This video demonstrates sectioning of low melting point agaraopse lmaembedded tissue and mounting nearnative sections. Animal husbandry and rapid dissection of the adult zebrafish heart. Current methods of mounting zebrafish embryos and larvae for imaging consist mainly of mounting in low percentage, low melting temperature agarose in a petri dish. Multilayer mounting enables longterm imaging of zebrafish. The journal of undergraduate neuroscience education june.
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