Zebrafish embryos methods

Verduzco, D. Amatruda, J. F. Analysis of cell proliferation, senescence, and cell death in zebrafish embryos. Methods Cell Biol. 2011,101,19-38. 

Mittelstadt SW, ffemenway CL, Craig MP, et al. Evaluation of zebrafish embryos as a model for assessing inhibition of hERG. / Pharmacol Toxicol Methods. 2008 57(2) 100-105. 

Zebrafish have emerged as a powerfiil model organism to study neutrophil chemotaxis and inflammation in vivo. Studies of neutrophil chemotaxis in animal models have previously been hampered both by the limited number of specimens available for analysis and by the need for invasive procedures to perform intravital microscopy. Due to the transparency and cell permeability of zebrafish embryos these limitations are circumvented, and the zebrafish system is amenable to both live time-lapse imaging of neutrophil chemotaxis and for screening of the effects of chemical compounds on the inflammatory response in vivo. Here, we describe methods to analyze neutrophil-directed migration toward wounds using both fixed embryos by myeloperoxidase activity assay, and live embryos by time-lapse microscopy. Further, methods are described for the evaluation of the effects of chemical compounds on neutrophil motility and the innate immune responses in zebrafish embryos. 

MARTINEZ-PARAMO S, PEREZ-CEREZALES S, ROBLES V, ANEL L and HERRAEZ M P (2008) Incorporation of antifreeze proteins into zebrafish embryos by a non-invasive method. Cryobiology, 56,216-222. 

Zebrafish Brachydanio/Danio rerio) embryos can be produced in large numbers and carry sufficient nutrients within the egg sack to allow development within micro plate volumes [52, 53]. Mutation frequency has been estimated indirectly using transgenic fish [54[ and UDS, comet, MNT and alkaline filter elution methods have been used effectively [55]. At present, there has been insufficient study of the model to understand predictivity of mammalian genotoxicity, and none of the methods has been demonstrated at throughputs sufficient for hit and lead screening. 

The methods presented here provide the basis for conducting zebrafish developmental toxicity research. Some fundamental procedures for the care and maintenance of a zebrafish colony for the purpose of collecting embryos has been provided, but greater detail and other procedures have been published elsewhere (12-14). While there are many very large zebrafish facilities and several very technically advanced zebrafish labs, one of the most attractive elements of the zebrafish (Fig. 1) as a model of embryonic development is how readily and inexpensively experiments can be performed. 

Procedures for maintenance of zebrafish colonies have been described by others in detail (17-19). Methods for maintaining a breeding colony and collecting embryos are included here. 

It is common to move adult zebrafish into breeding tanks when collections of embryos are desired. This type of breeding appears to be more stressful to the fish, but in most cases, zebrafish can be bred by this method approximately once per week. 

Chapin R, Augustine-Rauch K, Beyer B, Daston G, Finnell R, Flynn T, Hunter S, Mirkes P, O Shea KS, Piersma A, Sandler D, Vanparys P, Van Maele-Fabry G (2008) State of the art in developmental toxicity screening methods and a way forward a meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish. Birth Defects Res B Dev Reprod Toxicol 83 446-456... 

Some current limitations of RNAi should be mentioned in organisms such as Xmopus laevis (the African clawed toad) and Danio rerio (the zebrafish), RNAi has had limited success as a routine method, and only functions under certain circumstances. Here, the standard use of mor-pholinos (chemical modified antisense oli-gos) has routinely proven to be the method of choice, though more studies on RNAi are necessary to establish a routine use in cells and embryos of these vertebrates. 

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