Zebrafish embryos procedure

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. 

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. 

Embryos can then be used for experiments as described below or incubated and raised to add to the colony. Procedures for raising zebrafish have been described in detail previously elsewhere (17-19). 

Irrespective of how mutant embryos are produced, a similar protocol can be employed for the use of markers to detect mutant phenotypes. Zebrafish offer some unique alternatives in the detection of specific phenotypes. The ease with which embryos can be manipulated, the large brood size, optically clear embryos and reliability of procedures to detect mRNA or protein in fixed embryos allow the use of markers of specific developmental processes. Several markers may be used in combination. A typical protocol for marker-based screening is outlined below. Obviously, the stage of embryos fixed will vary between those markers used (see Note 8). 

Eix in a large volume (for this and subsequent procedures up to the hybridization step, use an excess, e.g., 20-fold by volume, of solution over embryo tissue) of MEMEA fix at 4°C for 4h-ovemight. Tissue can be stored at this point for a week. It is convenient to dechorionate zebrafish after fixation. 

This protocol works well on intact embryos up to at least mouse embryonic d 12, chick Hamburger and Hamilton (3) stage 24, and zebrafish to 72h (see also Note 7 for zebrafish modifications). For older embryos, it is likely that some block dissection is required prior to fixation to keep background levels low. Alternatively, some tissues, such as the neural tube, can be dissected after performing the procedure on intact older embryos. 

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