Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues. Zebrafish sample preparation for whole mount immunohistochemistry. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Embedding, serial sectioning and staining of zebrafish.
Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Zebrafish sample preparation for whole mount immunohistochemistry genetex. It is recommended that as incubations can be very long in whole mount staining, the antibody should be diluted in blocking buffer containing 0. Zebrafish ab strain were raised and maintained under standard conditions and staged as previously.
Whole mount immunofluorescence of zebrafish embryos. 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. 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. Zebrafish whole mount ihc of kidney tissue in embryos 72 hpf. Here youll find current best sellers in books, new releases in books, deals in books, kindle ebooks, audible audiobooks, and so much more. Fix freshly dissected tissue immunohistochemistry allows visualization of antigens usually proteins within an embryo. Chick or mouse embryo whole mount immunohistochemistry procedure based on protocols and information kindly provided by. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna. Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. The 5th edition is available in print and within the zfin protocol wiki. 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. Ihc staining protocol for whole mount samples abcam.
Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. Immunohistochemistry ihc protocolparaffin section protocol. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. 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. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. 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. We recommend following the zebra fish whole mount staining procedure provided. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. Whole mount immunohistochemistry in zebrafish embryos and. Professor anthony graham professor of developmental biology molecular neurobiology, medicine kings college london, uk dr.
Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. Representative imaging of wholemount immunohistochemistry of zebrafish. Histology and immunohistochemistry protocols from other online sources. Here we describe an ihc protocol for localizing the myosin heavy chain of zebrafish embryos at 12 and 35 days post fertilization dpf. Sequential immunofluorescence and immunohistochemistry on.
Whole mount staining of biotindextran injected zebrafish. 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. Useful tip for better zebrafish wholemount ihc staining. Ihc world is now offering full range of histology, ihc and tem services. In situ hybridization ish is a powerful technique for precise detection and localization of a specific nucleic acid sequence within a histologic section. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. Immunohistochemistry of wholemount mouse embryos were.
Electron microscopy tem digital image and analysis. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization. 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. 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. A guide for the laboratory use of zebrafish danio rerio 4th ed ed. Thisse lab in situ hybridization protocol 2010 update. In this chapter, a general protocol for wholemount immunofluorescent labeling 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. Whole mount in situ immunohistochemistry sciencedirect. The researcher should optimize the precise conditions for a particular assay. In situ hybridization ish nucleic acid retrieval principle, technique and protocol. General wholemount immunohistochemistry of zebrafish.
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. Prepare formalinfixed, paraffinembedded tissue sections step 18. 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. Flat mount preparation for observation and analysis of. Wholemount immunohistochemistry of the brain request pdf.
Here, we provide our protocol for whole mount fluorescence immunohistochemistry in zebrafish embryos and larvae. Therefore, it is important to know the characteristics of the antibody. However, there are relatively few zebrafish antibodies available. Our protocol can be accomplished in three days as outlined in figure 1. Staining wholemount embryos with pap protocols zfin. 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. 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 in zebrafish embryos. Treat with secondary antibody diluted with pbsbsadmso overnight at 4c or for 5 hr at. Wash twice in pbs, 5 minutes each, at room temperature. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Pdf wholemount immunohistochemistry for antif59 in zebrafish.
Displaying results 110 of 82 next results skin grafting in xenopus laevis. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf chapter pdf available january 2016 with 178 reads how we measure reads. Fluorescence microscopy for cilia in cultured cells and zebrafish. The following related protocols are also available. In particular, it is unsuited for routine screening applications. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data. Work from our laboratory has used these protocols to analyze kidney and heart development in zebrafish 38. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. A guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. Immunohistochemistry and in situ hybridization protocols nature protocols in situ hybridization protocols immunoportal fish protocols for drosophila.
Immunofluorescence protocol for confocal microscopy. Wash embryo with pbs twice for 1020 minutes each at rt. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. Please optimize the procedure since experimental conditions can vary for different tissue samples. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence.
Mattieu vermeron research fellow department of physiology, development and neuroscience. Zebrafish whole mount immunohistochemistry youtube. In this chapter, a general protocol for whole mount immunofluorescent labeling of zebrafish embryos and larvae is described. Both lna and mo based probes allow detection of robust mirna expression in whole mount zebrafish embryos of late developmental stages fig. W holemount immunohistochemistry ihc is a valuable tool for investigation of protein expression during zebrafish embryonic development. Xenopus wholemount immunohistochemistry protocol general information. Immunohistochemistry staining for frozen tissues acetonefixed ebioscience immunohistochemistry on schneider line 2 cells. Use of phospholipase a2 for antigen retrieval in zebrafish. Zebrafish whole mount highresolution double fluorescent in. In situ hybridization and immunohistochemistry are wellestablished and frequently used techniques to study the presence of mrna and protein.
We also provide information on how to couple jb4 embedding with wholemount rna in situ hybridization, immunofluorescence and gfp fluorescence. Staining slides for a discussion of antigfp and secondary antibodies. Spatial distribution of corticotropinreleasing factor. Our fixationpermeabilization procedure for immunofluorescence and. This protocol demonstrates sequential immunofluorescence and immunohistochemistry on cryosections from earlystage zebrafish embryos enabling precise colocalization analyses in. Mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital 2. However, their use in detecting lowabundance mirna. A simple whole mount staining protocol for bone and or cartilage in adults and larvae. Schultemerker from zebrafish book 5th edition fixation. Zebrafish whole mount highresolution double fluorescent in situ hybridization. Whole mount staining of zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. The whole mount in situ immunohistochemistry technique utilizes antibodies directly conjugated to fluorescent tags to visualize antibodybinding cells in live tissue explants. However, do not rock at high speed since the embryos are somewhat fragile. Incubate for 1 to 4 days on a gentle rotation devise at 4 oc.
Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific. This gives a clearer idea of where the target protein of interest is expressed within the. Although many immunohistochemistry protocols have been optimized for. Wholemount in situ hybridization of xenopus oocytes. Zebrafish whole mount highresolution double fluorescent. 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. General wholemount immunohistochemistry of zebrafish danio. The technique can also be easily adapted to detect anterograde and retrograde transport tracers. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform.
Oct 28, 2012 zebrafish sample preparation for whole mount immunohistochemistry genetex. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. Gentle fixation by freeze substitution gives excellent histological results with zebrafish embryos. This material is from the 4th edition of the zebrafish book. As with other immunohistochemical techniques, whole mount staining can be adapted to use many different antibodies enabling detection of specific cell types or other. In order to create a new protocol or add a comment, you must have a wiki account. 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. Commercially available antibodies will have a data sheet that will list the recommended. Immunohistochemistry ihc is a powerful method to determine localization of tissue components by the interaction of target antigens with labeled antibodies. 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. Zebrafish whole mount immunohistochemistry reagents. Clearing and staining for larval fish cartilage and bone.
Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. Twocolor fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. Whole mount immunohistochemistry and in situ hybridization of.
Immunohistochemistry ihc uses antibodies to detect cell and tissue proteins and provide semiquantitative data about target. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. Highresolution in situ hybridization to whole mount zebrafish embryos nature protocols whole mount in situ hybridization protocol. Not only are anatomical structures in zebrafish small, cells are also smaller. To circumvent these problems, this unit presents a routine whole. Wash slides in 100% alcohol twice for two minutes each. Immunohistochemistry protocol for frozen tissue download a pdf. Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Nuclei stained with propidium iodide are colored blue.
For icc experiments, cells must be attached to a microscope slide. Whole mount in situ hybridization and immunohistochemistry. Education guide immunohistochemical staining methods fifth. 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.
General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Wholemount immunohistochemistry for antif59 in zebrafish. Whole mount immunohistochemistry and in situ hybridization. In this chapter, i will describe the basic technique developed for wholemount labeling of zebrafish embryos and variations. A simple wholemount staining protocol for bone and or. Wholemount immunohistochemistry of the brain current. Whole mount immunohistochemistry in zebrafish embryos and larvae. Highresolution in situ hybridization to wholemount. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol.
In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is described. 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. Hydrolyze the probes to an average length of 150300 nucleotides following the protocol of cox et al. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by.
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. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope 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 is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues.
This incubation time will require some optimization depending on the antibody and also the size. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf. Here we describe an ihc protocol for localizing the myosin heavy. Whole mount edc fixation improves sensitivity and spatial resolution in mirna detection. 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. In addition, it is important to determine if the antibody will work with the sample that you wish to study.
Feel free to add new protocols to the appropriate section or add comments to any existing protocol. Achieving reliability and reproducibility of immunostains. Histology and immunohistochemistry online protocols. Method i frozen samples whole animal or dissected tissue. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. Deparaffinize slides in two changes of xylene for five minutes each. Fix for 1 hour, wash in pbs 1% triton then permeabilize the egg membrane in in ice cold acetone pbs for 8 minutes only. Histological methods protocols zfin community wiki. Typically, a primary antibody binds specifically to an antigen e. 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. Here, we present a protocol for fluorescent antibodymediated detection of. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. Basics and methods, a new book that aims to make the technique accessible to a wide audience.
This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. The books homepage helps you explore earths biggest bookstore without ever leaving the comfort of your couch. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Preparing paraffin tissue sections for immunostaining. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Immunohistochemistry is also an important complement to gene expression, as translation and transcription may not be concurrent.
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