The zebrafish larva is embedded in low melt agarose 1 % with the tail free to. Vegf inhibitor treatment embryos were mounted in 1% low melt agarose. With one of these coverslips, we filled a chaber of low melting point agarose gel including the zebrafish, removed all the excess agarose on top, and sealed it with a standard coverslip to prevent. A versatile mounting method for long term imaging of. Your new online msds binder is a place for you to store the material safety data sheets you need to deploy. Position of 10x images relative to 1 dpf zebrafish embryo. Due to their size and optical clarity, zebrafish embryos have long been appreciated for their usefulness in timelapse confocal microscopy. This corresponds to procedure steps 712, 14 and 15 from.
Eisen for more precise orientation of zebrafish embryos and to keep them from floating around during long term. 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. Vibratome sectioning for enhanced preservation of the. The cell nucleus serves as a mechanotransducer of tissue. Transfer a zebrafish embryo of choice to the agarose and take it up with a glass capillary inner diameter around 1 mm and a plunger. Agarose is a polysaccharide, generally extracted from certain red seaweed. Add low melting point agarose to 1x pbs and microwave until agarose is in solution. Actgene r9012le500g powder le agarose, multipurpose. Confocal imaging of live larval zebrafish for assessing. Agarose is highly biocompatible and possesses variable mechanical and diffusion properties. Quantitative microscopy of angiogenesis in zebrafish. A single method for cryofixation and correlative light, electron. To make gels with agarose concentration less than 2%. Sectioning lowmeltingpoint agarose lmaembedded tissue.
Agarose can be used as a gelling agent, to separate nucleic acids electrophoretically. Zebrafish husbandry, embryo collection, proteinase dechorionation, and embryo and larva maintenance were performed as described in the zebrafish book westerfield, m. Dna fragments of the equal size will take longer time to move through a low melting agarose gel compared to a standard agarose gel. We provide mounting protocols for the first days of zebrafish development for light sheet microscopy and other longterm imaging techniques. We have evaluated the development of zebrafish embryos in different mounting media and tested their suitability for spim. Multilayer mounting enables longterm imaging of zebrafish. The agar solution is added to the embryo when it is still a little bit hot and. At the desired stage 30 hpf, for example, take up the embryos in a pasteur pipette. This procedure requires certain important aspects that we have tested, which will optimize the outcome. Imaging subcellular structures in the living zebrafish. Gentle fixation by freeze substitution gives excellent.
Throughout this chapter, we defer to the zebrafish book westerfield 2000 on. Correlative light and electron microscopy of rare cell. The main properties of these agaroses are their low melting. Essentially, the process generates methoxylate groups from the basic agarose structure. A versatile mounting method for long term imaging of zebrafish. Once free of the agarose, vent should work in the fusion. Hyagarose le agarose is a low eeo, multipurpose, standard melting point agarose that yields high resolution sharp dna bands with high clarity and low background. Cells of interest in red primordial germ cells pgcs in this example, marked with asterisk. Introduction of the interactive atlas of zebrafish.
This can be avoided by using small volumes of low melting point agarose. 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. Low melting point lmp agarose gel preparation protocol. Gels, traditionally made with low melting agarose, are the most common way to mount samples for light sheet imaging. Cells free fulltext defective excitatoryinhibitory. The proteins may be separated by charge andor size isoelectric focusing agarose. Prepare the agarose to embed the fish by dissolving low melting agarose powder in danieaus buffer to a final concentration of 0. Set the plastic mold teeth down into the liquid agarose overlay, tapping to eliminate any bubbles. Two solutions of lowmelting point agarose lmpa were prepared in clean. Most likely, this is your first step in the purification process right after a restriction enzyme digest.
Beacon is created at the end of the yolk sac so that the resulting image is set around the trunk of the embryo. Agarose may be modified by hydroxyethylation, and this substitution reduces the number of intrastrand hydrogen bonds, resulting in lower melting and setting temperature than standard agaroses. B samples were embedded in 4% low melting point lmp agarose. Current methods of mounting zebrafish embryos and larvae for imaging consist mainly of mounting in low percentage, low melting temperature agarose. Melt 1% low melt agarose in a microwave or water bath. Search results for low melting agarose at sigmaaldrich.
Low melting or low gelling temperature agarose is produced by hydroxyethylation of agarose. I usually add approximately ul of low melt agarose in each well 48 well plate and let it. A single method for cryofixation and correlative light. How to mount zebrafish in agar zebrafish in the classroom. Agarose works well with living intact samples like zebra fish. Recovery of dna from low melting temperature agarose gels.
The embryos are held in wedgedshaped troughs made with a plastic mold in 1. For imaging, push out the part of the agarose which contains the embryo. Mechanical fixation techniques for processing and orienting delicate samples, such as the root of arabidopsis thaliana, for light or. Take care that the solution does not bubble over take. Methods and concepts in the life sciencesagarose gel. A device to hold zebrafish embryos during microinjection source. With one of these coverslips, we filled a chaber of low melting point agarose gel including the zebrafish, removed all. Capturing tissue repair in zebrafish larvae with time. Dissolve the low melting point agarose to a final concentration of 1. For example, morphogenesis of the zebrafish larva is investigated by embedding the fish in low melting agarose 9,10. Standardized mounting method of zebrafish embryos using a 3d.
Mounting the sample in low concentrations of agarose. The agarose tends to dry out, even though i add an extra drop of water at the start, whereas the coverslip tends to crush the fish after about 20 minutes of imaging. Arrows in b and d indicate the position of the vertical wall of the trough the trough is difficult to see in the photograph because the agarose has low contrast. For economy, normal agarose rather than low melting point agarose. Pipette one or two anesthetized embryos into a shallow glass depression slide. Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as dna or proteins in a matrix of agarose, one of the two main components of agar. A threeday old zebrafish embryo in lowmelting agarose prior to fast freezing. This video demonstrates sectioning of low melting point agaraopse lmaembedded tissue and mounting nearnative sections. Some heatinduced recombination in superficial structures can occur if embryos are exposed to larger volumes of low melting point agarose. A anesthetized larval zebrafish are placed into a methylcellulose pictured or lowmeltingpoint agarose solution before being loaded into the capillary housing. The earlier use of allover frosted slides has been superseded by slides precoated with agarose and dried.
Agarose, low gelling temperature 2hydroxyethyl agarose. The exact temperature is determined by the degree of substitution, many available low melting point lmp agarose. A versatile mounting method for long term imaging of zebrafish development. It is a linear polymer made up of the repeating unit of agarobiose, which is a disaccharide made up of dgalactose and 3,6anhydrolgalactopyranose. Embryos can also be dechorionated and embedded in agarose e. I had good results with low melting point agarose, as suggested above.
Low melting lm agaroses are the result of a derivatization process by organic synthesis. Heat it in the microwave for about 15 seconds watch it to make sure it doesnt bubble over. If pcr generated errors are of concern then each pcr generated fragment can be extracted from the low melt agarose and etoh precipitated. Also the petridish with the low melting agarose was stored at 4 c.
If agarose is dropped onto a plain glass microscope slide, the gel is likely to detach during lysis. Agar is also a plant abstract dissolved in fish water to ultimately make a gel that holds the embryo. Eisen for more precise orientation of zebrafish embryos and to keep them from floating around during long term observations, embed them in agar. Zebrafish danio rerio adults and embryos were kept at 28. Lowcost silicone imaging casts for zebrafish embryos and. The main properties of these agaroses are their low melting and gelling temperatures when compared with standard agaroses. Low melting agarose tbe buffer dna fragment phenol chloroform isoamyl alcohol note 1. How best to immobilise zebrafish larvae for imaging. It is usually used for the isolation of separated dna fragments.
Fine structural preservation of hpf zebrafish embryos after rapid fs. Freeze substitution is a previously described technique for the gentle fixation and dehydration of tissue. Let the agarose solidify and keep the embryo in the capillary submerged in fish medium. Intravital imaging of metastasis in adult zebrafish bmc. To melt the agarose, simply by heating the slurry in a boiling water bath, bring the solution to a boil and allow it to boil for 510 minutes stirring continuously, until the agarose dissolves completely. Low melting point lmp agarose gel preparation protocol 1. In rather low concentrations 0,5% to 1,0% it is translucent, cheap, chemically inert and has a refractive index close to water. Agarose powder, high pure quick dissolve agarose for dna. Weinberg this is an easy method for holding embryos while injecting dna, lineage tracer dyes, etc.
74 1230 1167 665 329 83 1152 1278 586 474 1518 999 1011 800 1587 96 513 371 806 959 662 1403 227 1151 1463 964 1586 1216 1075 1279 1311 297 827 1162 1281