To facilitate the required incubation steps, whole tissues must be cut into ultra thin 510 mm sections or cut into smaller pieces for whole mount ihc. Not only are anatomical structures in zebrafish small, cells are also smaller. A guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. Whole mount immunohistochemistry in zebrafish embryos and. Schultemerker from zebrafish book 5th edition fixation. Incubate for 1 to 4 days on a gentle rotation devise at 4 oc. We recommend following the zebra fish whole mount staining procedure provided. 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. Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. Wholemount immunohistochemistry of the brain current. 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. W holemount immunohistochemistry ihc is a valuable tool for investigation of protein expression during zebrafish embryonic development. Typically, a primary antibody binds specifically to an antigen e.
Ihc world is now offering full range of histology, ihc and tem services. Whole mount fluorescent immunohistochemistry the advantage of using fluorescence to stain whole mount sections is that confocal microscopy can be used to section through the larger embryo or tissue sample without having to manually section onto slides. Video articles in jove about whole mount immunohistochemistry include flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization, visualization and analysis of pharyngeal arch arteries using wholemount immunohistochemistry and 3d reconstruction, functional and morphological assessment of diaphragm innervation by. Immunohistochemistry ihc is a powerful method to determine localization of tissue components by the interaction of target antigens with labeled antibodies. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. A simple whole mount staining protocol for bone and or cartilage in adults and larvae. Fix freshly dissected tissue immunohistochemistry allows visualization of antigens usually proteins within an embryo. Oct 28, 2012 zebrafish sample preparation for whole mount immunohistochemistry genetex. This incubation time will require some optimization depending on the antibody and also the size. Zebrafish whole mount ihc of kidney tissue in embryos 72 hpf. Hydrolyze the probes to an average length of 150300 nucleotides following the protocol of cox et al. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf.
Use of phospholipase a2 for antigen retrieval in zebrafish. The researcher should optimize the precise conditions for a particular assay. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. Method i frozen samples whole animal or dissected tissue. Whole mount edc fixation improves sensitivity and spatial resolution in mirna detection. Preparing paraffin tissue sections for immunostaining. Commercially available antibodies will have a data sheet that will list the recommended. Immunofluorescence microscopy is one of the numerous ihc methods that may be used to assess both the localization and endogenous levels of proteins of interest. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. Wash slides in 100% alcohol twice for two minutes each. Basics and methods, a new book that aims to make the technique accessible to a wide audience.
This is an openaccess article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Zebrafish whole mount highresolution double fluorescent in situ hybridization. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is described. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Whole mount in situ immunohistochemistry sciencedirect. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact. Chick or mouse embryo whole mount immunohistochemistry procedure based on protocols and information kindly provided by. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. Depending on the thickness of the bones we did it with whole mouse heads to cut the paranasal sinuses and the cribriform plate within the bony head, with. In this chapter, i will describe the basic technique developed for wholemount labeling of zebrafish embryos and variations. Zebrafish ab strain were raised and maintained under standard conditions and staged as previously. Gentle fixation by freeze substitution gives excellent histological results with zebrafish embryos. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals.
Whole mount staining of biotindextran injected embryos photoconversion of fluorescently labeled profiles for em a simple whole mount staining protocol for bone and or cartilage in adults and larvae. Our fixationpermeabilization procedure for immunofluorescence and. For icc experiments, cells must be attached to a microscope slide. Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. Treat with secondary antibody diluted with pbsbsadmso overnight at 4c or for 5 hr at. Staining slides for a discussion of antigfp and secondary antibodies. Useful tip for better zebrafish wholemount ihc staining. Protocols, pitfalls, and tips sowoon kim jin roh chansik park department of pathology, asan medical center, university of ulsan college of medicine, seoul, korea immunohistochemistry ihc is an important auxiliary method for pathologists in routine diagnostic. Immunofluorescence protocol for confocal microscopy. 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. Electron microscopy tem digital image and analysis. Spatial distribution of corticotropinreleasing factor. Achieving reliability and reproducibility of immunostains.
Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. Both lna and mo based probes allow detection of robust mirna expression in whole mount zebrafish embryos of late developmental stages fig. Professor anthony graham professor of developmental biology molecular neurobiology, medicine kings college london, uk dr. Zebrafish whole mount highresolution double fluorescent in. In order to create a new protocol or add a comment, you must have a wiki account. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. Here we describe an ihc protocol for localizing the myosin heavy chain of zebrafish embryos at 12 and 35 days post fertilization dpf. In situ hybridization and immunohistochemistry are wellestablished and frequently used techniques to study the presence of mrna and protein. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Xenopus wholemount immunohistochemistry protocol general information.
Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Mattieu vermeron research fellow department of physiology, development and neuroscience. Immunohistochemistry ihc uses antibodies to detect cell and tissue proteins and provide semiquantitative data about target. Nuclei stained with propidium iodide are colored blue. 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. Immunohistochemistry is also an important complement to gene expression, as translation and transcription may not be concurrent. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna. Wash twice in pbs, 5 minutes each, at room temperature. A simple wholemount staining protocol for bone and or. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence. Histological methods protocols zfin community wiki. Zebrafish whole mount immunohistochemistry reagents. The books homepage helps you explore earths biggest bookstore without ever leaving the comfort of your couch.
Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. Here, we provide our protocol for whole mount fluorescence immunohistochemistry in zebrafish embryos and larvae. Twocolor fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. The 5th edition is available in print and within the zfin protocol wiki. Pdf wholemount immunohistochemistry for antif59 in zebrafish. Histology and immunohistochemistry protocols from other online sources. The following related protocols are also available. Zebrafish whole mount highresolution double fluorescent. Thisse lab in situ hybridization protocol 2010 update. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides. Here, we describe a protocol for whole mount immunohistochemistry and in situ hybridization that can be applied to all stages of zebrafish development and dissected bony parts.
If an antibody has been used successfully on cryosections this does not include paraffinembedded sections, then the antibody should work for a whole mount embryo. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. Wholemount in situ hybridization of xenopus oocytes. In addition, it is important to determine if the antibody will work with the sample that you wish to study. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data. This gives a clearer idea of where the target protein of interest is expressed within the. Highresolution in situ hybridization to wholemount. Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific. Staining wholemount embryos with pap protocols zfin. This material is from the 4th edition of the zebrafish book. Wholemount immunohistochemistry of the brain request pdf. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish.
Flat mount preparation for observation and analysis of. Whole mount immunohistochemistry and in situ hybridization of. Ihc staining protocol for whole mount samples abcam. Mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital 2. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Zebrafish whole mount immunohistochemistry youtube. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Education guide immunohistochemical staining methods fifth. We also provide information on how to couple jb4 embedding with wholemount rna in situ hybridization, immunofluorescence and gfp fluorescence.
To circumvent these problems, this unit presents a routine whole mount immunocytochemistry protocol that can be used to map many antigenic distributions in the developing and adult brain. Please optimize the procedure since experimental conditions can vary for different tissue samples. Displaying results 110 of 82 next results skin grafting in xenopus laevis. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol. Immunohistochemistry and in situ hybridization protocols nature protocols in situ hybridization protocols immunoportal fish protocols for drosophila.
Embedding, serial sectioning and staining of zebrafish. Here, we present a protocol for fluorescent antibodymediated detection of. In situ hybridization ish nucleic acid retrieval principle, technique and protocol. Although whole mount immunohistochemistry is commonly used with embryo or organ specimens in drosophila and xenopus, its use in murine research has been limited to the study of embryogenesis 11. Our protocol can be accomplished in three days as outlined in figure 1. Wholemount immunohistochemistry for antif59 in zebrafish. It is recommended that as incubations can be very long in whole mount staining, the antibody should be diluted in blocking buffer containing 0.
Whole mount immunohistochemistry in zebrafish embryos. Highresolution in situ hybridization to whole mount zebrafish embryos nature protocols whole mount in situ hybridization protocol. As with other immunohistochemical techniques, whole mount staining can be adapted to use many different antibodies enabling detection of specific cell types or other. General wholemount immunohistochemistry of zebrafish. Wholemount 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. Video articles in jove about whole mount immunohistochemistry include flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization, visualization and analysis of pharyngeal arch arteries using whole mount immunohistochemistry and 3d reconstruction, functional and morphological assessment of diaphragm innervation by. Whole mount staining of biotindextran injected zebrafish. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from earlystage zebrafish embryos enabling precise colocalization analyses in. Whole mount immunofluorescence of zebrafish embryos. Representative imaging of wholemount immunohistochemistry of zebrafish.
For in situ hybridization, here, we outline how to generate rna probes, conduct whole mount in situ hybridization for larvae, and list rna probes that label different stages of schwann cell development in zebrafish. Fluorescence microscopy for cilia in cultured cells and zebrafish. In particular, it is unsuited for routine screening applications. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf chapter pdf available january 2016 with 178 reads how we measure reads.
Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. In this chapter, a general protocol for whole mount immunofluorescent labeling of zebrafish embryos and larvae is described. Histology and immunohistochemistry online protocols. Therefore, it is important to know the characteristics of the antibody. To circumvent these problems, this unit presents a routine whole. The whole mount in situ immunohistochemistry technique utilizes antibodies directly conjugated to fluorescent tags to visualize antibodybinding cells in live tissue explants. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. However, there are relatively few zebrafish antibodies available. Analysis of protein expression and localization by zebrafish immunostaining ihc and if, in sections or whole mount preparations ihcwm, is easy to perform with zebrafish research primary antibodies. Whole mount immunohistochemistry and in situ hybridization. Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues.
Immunohistochemistry of wholemount mouse embryos were. 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 fig. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Deparaffinize slides in two changes of xylene for five minutes each. Although many immunohistochemistry protocols have been optimized for.
A guide for the laboratory use of zebrafish danio rerio 4th ed ed. However, their use in detecting lowabundance mirna. Here we describe an ihc protocol for localizing the myosin heavy. Wash embryo with pbs twice for 1020 minutes each at rt. Whole mount staining of zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. However, do not rock at high speed since the embryos are somewhat fragile. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Prepare formalinfixed, paraffinembedded tissue sections step 18. Place a metal cryostat chuck on top of the dry iceisopentane slurry and then mount piece of tissue on top of the chuck and cover it with oct. Immunohistochemistry staining for frozen tissues acetonefixed ebioscience immunohistochemistry on schneider line 2 cells. Sequential immunofluorescence and immunohistochemistry on. General wholemount immunohistochemistry of zebrafish danio. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community.
Immunohistochemistry protocol for frozen tissue download a pdf. Whole mount immunohistochemistry in zebrafish embryos and larvae. The technique can also be easily adapted to detect anterograde and retrograde transport tracers. Here youll find current best sellers in books, new releases in books, deals in books, kindle ebooks, audible audiobooks, and so much more.
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. Zebrafish sample preparation for whole mount immunohistochemistry genetex. Clearing and staining for larval fish cartilage and bone. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Whole mount in situ hybridization and immunohistochemistry. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization.
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