Interactions with iridophores and the tissue environment. The first step in almost every investigation of skeletal phenotypes is analysis of wholemount skeletal preparations. Multiplex fluorescence in situ hybridization fish enables you to assay multiple targets and visualize colocalized signals in a single specimen. Genomewide chemical mutagenesis screens in the zebrafish. The visualization of gene expression throughout the larvae again is possible due to the optical clarity of the zebrafish tissues, which allows successful penetration for light microscopy. Native to south asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio and thus often called a tropical fish although both tropical and subtropical. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3.
Maximum projection of dorsal view of whole mount zebrafish top panels. In the past two decades, zebrafish has become one of the preferred in vivo model organisms for studying diverse processes like embryogenesis 8, 9, autophagy, behaviour 11, 12 and sleep. Wholemount immunofluorescent staining facilitates the profiling of protein expression patterns within diverse and complex tissues. Whole mount staining of biotindextran injected zebrafish embryos photoconversion of. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Todays deals your gift cards help whole foods registry sell disability customer support. Unfortunately, tca is a fairly strong acid and will corrode the tissue processor. Twocolor fluorescent in situ hybridization in the embryonic zebrafish brain using. Using spectrally distinct fluorophore labels for each hybridization probe, this approach gives you the power to resolve several genetic elements or multiple gene expression patterns through multicolor visual display.
This technique can be used to determine, with the presence or absence of a fluorescent signal, whether specific genetic elements exist in a sample. The znp1 antibody will specifically bind to the primary motor neurons. The 5th edition is available in print and within the zfin protocol wiki. Useful tip for better zebrafish wholemount ihc staining. In performing flat mounts on wish zebrafish embryos with the protocol described in parts 15, the initial embryo fixation and final staining. Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development will need to be much longer than normal immunocytochemistry. Transparency is a unique attribute of zebrafish that permits direct assessment of drug effects on the nervous system using whole mount antibody immunostaining and histochemistry.
Embedding, serial sectioning and staining of zebrafish. Although you can stain both cartilage and bone in the same specimen, you need to be aware that bone staining can mask cartilage staining, for instance when perichondral ossification takes place around a cartilage template. We created wounds at the tip of zebrafish embryo tails and perform immune staining for. Whole mount immunohistochemistry in zebrafish embryos and. One for alla highly efficient and versatile method for fluorescent. The intestine was removed from fish of different ages and examined by wholemount in situ hybridization. Whole mount antibody staining of zebrafish embryos for. Book cover trout fish book sleeve adhesivefree pu leather journal cover stylish trade cover large size 9x 11 students bookworm gift bookish gift. Whole mount staining of zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. In summary, development of the cranial neuronal and glial lineages that are derived from neural crest and ectodermal placodes depends on tfap2a activity. Not only are anatomical structures in zebrafish small, cells are also smaller.
General whole mount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Fix for 1 hour, wash in pbs 1% triton then permeabilize the egg membrane in in ice cold acetone pbs for 8 minutes only. Flat mount preparation for observation and analysis of. Fluorescence in situ hybridization fish thermo fisher. Zebrafish whole mount immunohistochemistry reagents. The whole mount staining of various mouse tissues provides an opportunity to study the entire vasculature including whole vessel width, vascular morphogenesis, and valve formations. Analysis of apoptosis in zebrafish embryos by wholemount. Zebra fish whole mount in situ hybridization protocols roe lab products and services. Larvae at 6 dph were tested in groups of fish in a clear acrylic plate. This protocol describes ish of digoxigeninlabeled antisense rna. This article is from journal of visualized experiments. Whole mount immunohistochemistry in zebrafish embryos. In recent years, the zebrafish embryo has emerged as a popular model to.
For immunostaining with the laminin 1 antibody sigma l9393 1. Clinical signs include lethargy, anorexia, and emaciation. Analyzing wound healing in zebrafish embryos february 11. Whole mount staining of zebrafish embryos, now commonly used, requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilized. Whole mount antibody staining of zebra fish larvae modified protocol from stefan schultemerker. In the zebrafish, neutrophils were negative and the.
In situ hybridization ish is a type of hybridization that uses a labeled complementary dna, rna or modified nucleic acids strand i. In wholemount ihc, the distribution of an antigen can be. To assess pharmacological effects of drugs on the optic nerves, motor neurons, and dopaminergic neurons, we performed whole mount immunostaining and visualized different neuronal. Pairwise breeding of individual or natural cross fish.
The protocol is a modified version of the standard in situ. Zebrafish as a model vertebrate for investigating chemical. Wholemount immunostaining methods to study the blood and. Chapter 9 molecular methods whole mount in situ hybridization source. Wholemount skeletal staining permits evaluation of the shapes and sizes of. Zebra mussels and aquatic nuisance species is a thorough, wellreferenced, and illustrated look at the growing problem of nonindigenous species pollution. Zebras fish book all steps in the procedure are carried out at room temperature. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. Antibody staining and whole mount in situ hybridization. We show that the teleost fish, fugu and zebrafish, have two ribeye genes, ribeye a and ribeye b. Clinical and macroscopic changes associated with mycobacterium may be variable, dependent upon the site and extent of the infection, and on the chronicity of the infection. Whole mount antibody staining of zebrafish embryos reagents paraformaldehyde 10x pbs triton x100 primary antibody alexafluor conjugated secondary antibody bsa dmso optional methanol normal sheep serum heat inactivated at 56 c for 30 minutes methylcellulose glycerol permount fisher sp15100 procedure fixation and storage of embryos. The protocol is a modified version of the standard in situ hybridization. Professor anthony graham professor of developmental biology molecular neurobiology, medicine kings college, london, uk whole mount staining of zebrafish embryos, now commonly used, requires extra steps to fix and permeabilize to.
This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Additionally data quantification of lymphaticblood vasculature is often most accurate with. Whole mount antibody staining of zebrafish embryos for markers of segmentation. The overall goal of this method is to identify the relative abundance and location of apoptotic cells in zebrafish from the 4cell stage to 32 hrpostfertilization hpf. Dechorionate 26 hr embryos pharyngula stage carefully with two fine forceps. Effects of lead on neurogenesis during zebrafish embryonic brain development. The epub format uses ebook readers, which have several ease of reading. Effects of lead on neurogenesis during zebrafish embryonic. Fish is a molecular technique that is often used to identify and enumerate specific microbial groups. Flat mount preparation for observation and analysis of zebrafish. Here, we present a highresolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes.
Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Effects of copper oxide nanoparticles on developing zebrafish embryos and larvae yan sun, gong zhang, zizi he, yajie wang, jianlin cui, yuhao li department of pathology, key laboratory of tumor microenvironment and neurovascular regulation, nankai university school of medicine, tianjin, peoples republic of china abstract. Flat mount preparation for observation and analysis of zebrafish embryo specimens. The zebrafish danio rerio is a freshwater fish belonging to the minnow family of the order cypriniformes. Zebrafish are particularly amenable to this technique and it is possible to localize proteins both in whole embryos and larvae, as well as sectioned material. Wholemount tunel terminal deoxynucleotide transferase mediated dutp nickend labeling staining. I wanted to ask if anyone knows how to bleach the pigment of 2dpf zebrafish embryos post whole mount in situ hybridization it was not. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. The fish must be thoroughly rinsed to remove any tca before the fish can be processed. General wholemount immunohistochemistry of zebrafish danio. Wholemount in situ hybridization revealed that ribeye a is expressed in tissues containing synaptic ribbons, including the pineal gland, inner ear, and retina. Ribeye is the only known protein specific to synaptic ribbon, but its function is unclear. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ.
Representative imaging of wholemount immunohistochemistry of zebrafish danio. Whole mount staining of biotindextran injected zebrafish embryos. Fix the fish in 10% neutral buffered formalin for a few hours to one day depending on the size of the specimen. The in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Abstract wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The full recovery of the wound normally requires 35 days and recapitulates the process of tail fin regeneration in adult fish. The zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Copper oxide nanoparticles cuo nps are used for a variety of. Therefore, samples should be equilibrated in 75% glycerol after staining for approximately 15 minutes again, when equilibrated, the sample should sink. The embryo can be imaged at this stage, or mounted whole in the glycerol on a slide. Feel free to add new protocols to the appropriate section or add comments to any existing protocol.
Hello im shelly, a phd student at gothilfs lab from tel aviv university. Wholemount larval staining can be performed rather than first having to dissect the tissue or stain sections. Whole mount immunohistochemistry in zebrafish embryos and larvae. Visualizing multiciliated cells in the zebrafish through a combined. Hardcopies of the 4th edition of the zebrafish book can be obtained for a nominal fee from the zebrafish international resource center, 5274. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. Wholemount staining of biotindextran injected zebrafish embryos. Ive been busy trying to stain the eyes of my zebra fish, but still with little luck.
Immunostaining of dissected zebrafish embryonic heart. Patches consisting of cells staining with both probes were. Mpo activity has been used as a marker of zebrafish neutrophils, although weak staining has also been observed in. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with. Photoconversion of fluorescently labeled profiles for em. Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. We obtained similar results analyzing the ret expression pattern by wholemount in situ hybridization data not shown. Fix anaesthetized larvae in 4% pfa for 4 hours or overnight at 4. Effects of copper oxide nanoparticles on developing.
The whole mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. Zebrafish whole mount highresolution double fluorescent. In addition, the development of somites in a 24 hour old zebrafish will also be. In situ hybridization staining buffer 100 mm tris ph 9.
A guide for the laboratory use of zebrafish danio brachydanio rerio. In this chapter, i will describe the basic technique developed for wholemount labeling of. Methodologyprincipal finding here we present a novel method to. Zebrafish whole mount immunohistochemistry youtube.
Eviscerate the specimen removing internal organs being careful not. Zebrafish mutants identify an essential role for laminins. A guide for the laboratory use of zebrafish danio rerio. Wholemount in situ hybridization assays were performed according to the method of schultemerker et al. Modified protocol for staining sectioned tissue that is mounted on slides. Wholemount larvae were stained with myeloperoxidase kit sigma for histochemical identification of neutrophils. Wholemount staining of biotindextran injected zebrafish embryos photoconversion of. This material is from the 4th edition of the zebrafish book. Fluorescence in situ hybridization fish is a molecular cytogenetic technique that uses fluorescent probes that bind to only those parts of a nucleic acid sequence with a high degree of sequence complementarity. Whole mount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. My goal is to dye the retinas and their resulting optic. Not all iridophore reflecting platelets are visible and platelets that are apparent may not precisely delineate cell bodies and processes. A simple wholemount staining protocol for bone and or.
We currently offer a continuously expanding product list for research in zebrafish. If the signal is strongly diminished after the methanol treatment, one can use the nonmethanol protocol, or test alternative antibodies against the target protein. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna sequences on chromosomes. We recommend following the zebra fish whole mount staining procedure provided. Here we describe how to perform whole mount immunofluorescence in early zebrafish embryos to detect cells with activated caspase 3 referred to hereafter as the casp3 assay. Neurotoxicity assessment using zebrafish sciencedirect. Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues.
Recently, zebrafish has sparked interest in other branches of the biomedical research. General wholemount immunohistochemistry of zebrafish. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. About 100200 embryos are readily available every week from a single pair of adult fish. Thanks to the application of antibodies on whole mounted instead.
Fish may exhibit ulcers, hemorrhage or hyperemia around the head similar to gram negative septicemia, raised scales, frayed fins, and pallor of the skin or. Immunohistochemistry ihc is a powerful research tool to localize specific antigens in wholemount or tissue sections of embryos with labeled antibodies based on antigenantibody interactions. Zebrafish danio rerio is a fresh water fish that inhabits rivers in india, pakistan and other places in asia. The following is a staining protocol perfected by karen larison at zirc that we. The analysis of activated caspase 3 by wholemount immunofluorescence in zebrafish embryos reveals stage and tissuespecific localization of cells specifically undergoing apoptosis.
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