The dechorinated zebrafish embryo developmental toxicity assay. Cryopreservation and in vitro fertilization ivf protocols. Some protocols require a few additional steps for embryo maintenance. Formalin or other aldehyde fixation forms protein crosslinks that mask the antigenic sites in tissue specimens, thereby giving weak or false negative staining for immunohistochemical detection of certain proteins. The laboratory protocol that was adapted from harvey et al. The red nucleus nucleus ruber is an important neuronal structure in the midbrain tegmentum, and it controls motor coordination regulating in particular the forelimbs during locomotion. After 1014 days, the paramecia are ready to feed to the fish larvae. Zebrafish developmental toxicity assay oxford academic journals. Analysis of cell proliferation, senescence and cell death. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon.
In this study, we employed zebrafish injected with morpholinos against genes involved in the pld, including sec63, prkcsh, and pkd1a. A method for the assessment of dna damage in individual. To each bowl add 89 grains of of boiled wheat and 8 ml from a paramecia seed culture. Chemicals should be dissolved in fish water if possible see. Remove embryos from their chorions by immersing them in 1 mgml pronase for 5 minutes, room temperature, in a petri dish. Wholemount in situ hybridization of bmp7a in zebrafish embryos, after treatment with bmp inhibitors andor ginger10g from 10 to 48 hpf. In the case that this is not possible, the solvent of choice for exposure utilizing the embryonic zebrafish is dimethyl sulfoxide dmso see. Removing embryos from their chorions protocols zfin. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Pronase is the name given to a group of proteolytic enzymes that are produced in the culture supernatant of streptomyces griseus k1 1, 2, 3. For the first three days of life, zebrafish embryos develop inside of a chorion, which can impede manipulation of the embryo. Product description type unit quantity academic price book.
The pronase based solution is designed to break the protein crosslinks, therefore unmask the antigens and epitopes in formalin. The protocol described herein uses an antibody that specifically binds the cleaved, heterotetrameric form of caspase 3, but not the inactive caspase 3 dimer. The in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Cultures remain useable and healthy for a month or more. Embryos younger than 24 h require shorter exposure to pronase see removing embryos from their chorions, chapter 4. The journal will serve as a forum for papers discussing research on comparative genomics and evolution, the genetic analysis of embryogenesis and disease and the. Representation of an 8h zebrafish embryo, showing the. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Embryos younger than 24 hpf require shorter exposure to pronase see removing embryos from their chorions. The embryonic zebrafish model offers the power of wholeanimal. Basic protocols for zebrafish cell lines article pdf available in methods in molecular biology 362. Western blots of zebrafish embryos 090511071927884 s. Dechorionating zebrafish embryos stanford university. Pronase is known to contain at least ten proteolytic components.
Pronase protease, streptomyces griseus sigmaaldrich. The chorion can be removed by treatment with the proteolytic enzyme, pronase. It is also used in histochemistry and cell culture for tissue dissociation in conjunction with collagenase and trypsin, and for the production of glycopeptides from purified glycoproteins. Remove as much egg water as possible and add pronase on them 1 mgml in egg water and swirl the petri dish. Evaluation of embryotoxicity using the zebrafish model ncbi nih. The zebrafish information network zfin is an online database that collects, curates, and disseminates information about zebrafish biology. Take a petri dish coated with 1% agarose gel and transfer the embryos to be dechorionated in there. Identification of the zebrafish red nucleus using wheat. Dechorionation of zebrafish embryos with pronase for. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. The zebrafish is an ideal organism for small molecule studies.
It is not necessary to let pronase selfdigest prior to use. Preparation of medium for zebrafish embryios e3 medium filed under. Remove as much egg water as possible and add pronase on them 1 mgml in egg. Danio rerio, or zebrafish, are small fish that are making a big splash in biomedical research. Part i of this book takes advantage of the highthroughput nature of larval models to offer protocols for research requiring high output, easily manipulated screens.
Pretreatment with a dilute solution of pronase 2 mgml in embryo medium, 1 min, 28. Here youll find current best sellers in books, new releases in books, deals in books, kindle ebooks, audible audiobooks, and so much more. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. This protocol describes ish of digoxigeninlabeled antisense rna. Application of the existing deyolking protocol 16 to zebrafish early embryos requires a. Proteomics analysis of early developmental stages of zebrafish. Generating chimeric zebrafish embryos by transplantation. Zebrafish embryos 100 embryos from different stages were dechorionated in eppendorf tube either with pronase 50. Stack the finger bowls 6 or 8 high, cover, and store at 28. The zebrafish as a model to study polycystic liver disease. Remove embryos from their chorions in batches of 100 by placing in 1 mgml of pronase and swirling occasionally 510 minutes for 24 h. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. Pour each test solution into a 50 ml reagent reservoir, which will fit a multichannel pipette.
Embryos are easily treatable by waterborne exposure. If the embryos are chased for varying amounts of time after the brdu pulse, then fixed and stained for both brdu and ph3 section b, the transit of cells from s. Pdf a brief protocol for highresolution whole mount in. A stepbystep guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage. Cryopreservation and in vitro fertilization ivf protocols embryo surface sanitation egg bleaching protocol fish feeding protocols. For dechorionation, add 9 ml of embryo medium containing 0. Zebrafish are increasingly being used as a model system for understanding the genetic and neuronal basis of behavior. Embryos to be raised should be bleached during gastrula stages will still hatch on their own or by day 1 you will need to remove chorions by treating with pronase or manually of development follows. This development is catalyzed by a unique combination of strengths, including the ability to isolate mutants, make transgenic animals with different cell types labeled by fluorescent markers, and record from neurons in the. Zebrafish protocols for neurobehavioral research addresses protocols for both larval and adult models, written by the leading experts in the field of zebrafish research. Historically, zfin hosted the zirc site and databases.
Evaluation of embryotoxicity using the zebrafish model. In the polycystic liver diseases pld, genetic defects initiate the formation of cysts in the liver and kidney. Transfer them to a clean beaker filled with 10% hanks saline and rinse several times with clean 10% hanks. Small molecule screening in the zebrafish sciencedirect. Westerfield from zebrafish book 5th edition some observations of zebrafish development can be made directly through the chorion, however for most procedures it is better to remove the chorion. Bleaching zebrafish embryos reagents and supplies e3 medium quantities for 5 l of 60x stock 5 mm nacl 86 g 0. The standard fish embryo toxicity test fet with luviquat hm 522 was conducted in three replicates according to din 38415t6 and iso 15088 din, 2001, iso, 2007 as described in lammer et al. Finish dechorionation by gentle trituration using a pasteur pipette. One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos.
A guide for the laboratory use of zebrafish danio rerio, eugene, university of. Zirc protocols zirc public wiki zebrafish international. Deyolk and dechorionate zebrafish embryos ms utils. Zebrafish lay hundreds of eggs that develop externally, allowing scientists to perform genetic manipulations and monitor early phenotypes in a complex organism. Stock 100 mm phenylmethylsulfonylfluoride in isopropanol. Dechorionation as a tool to improve the fish embryo. One of the advantages of the casp3 assay is the ability to visualize the spatial distribution of apoptotic cells within the context of the whole embryo. The use of pronase e as described in the present paper, reduced the background dna damage level by a factor of 5. The following protocol is designed to label a fraction of proliferating cells in zebrafish embryos, to allow comparison of the replication fraction of different embryos figure 1 b. Cuttingedge and timely, zebrafish neurobehavioral protocols serves as an excellent compilation epitomizing the growing potential of zebrafish in modern scientific. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to.
Extraction of proteins from zebrafish embryos for sdsgel. Protein extraction with deyolk for western blot protocol. Preparation of medium for zebrafish embryios e3 medium. This how to book will be a useful practical guide for zebrafish researchers and will complement another related book from this series, zebrafish models in neurobehavioral research. Today, the two web sites still appear very similar, because they are closely related in their mission to serve the research community. Analysis of apoptosis in zebrafish embryos by wholemount. Dechorionating zebrafish embryos reagents and supplies e3 medium quantities for 5 l of 60x stock 5 mm nacl 86 g 0. Zebrafish has emerged as an informative animal model to study the biological impact and molecular mechanisms of hypoxia.
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