In situ hybridization ish nucleic acid retrieval principle, technique and. Immunohistochemistry ihc uses antibodies to detect the location of proteins and other antigens in tissue sections. Here we present a novel method to efficiently retrieve the antigen in. Wildtype zebra fish tu strain were obtained from zirc university of oregon, eugene, usa. In addition, it is important to determine if the antibody will work with the sample that you wish to study. This protocol describes ish of digoxigeninlabeled antisense rna probes to whole mount. Whole mount immunohistochemistry in zebrafish embryos. Flat mount preparation for observation and analysis of. 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. Wash embryo with pbs twice for 1020 minutes each at rt.
General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19. Mount the coverslips on a slide in glycerolbased mounting medium. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. Rna fish on whole mount blastocyst rna fish on whole mount 6. Wholemount in situ hybridization wish is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. In situ hybridization ish nucleic acid retrieval principle, technique and protocol. Wholemount staining of biotindextran injected embryos photoconversion of fluorescently labeled profiles for em a simple wholemount staining protocol for bone and or cartilage in adults and larvae. Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides.
Different antibodies and tissues may require changes to this procedure. This is an official page of the university of massachusetts medical school. Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. This concise yet comprehensive guide to the methods and protocols of immunohistochemistry covers established techniques and current developments in the field such as the use of epitope tags, multiple immunolabeling and diagnostic immunohistochemistry. Zebrafish are a tropical, freshwater vertebrate species belonging to the family cyprinidae. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. Deparaffinize slides in two changes of xylene for five minutes each. This guide highlights critical steps in the ihc protocol and demonstrates how protocol changes can affect the final. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna sequences on chromosomes. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. Immunohistochemistry ihc is a common approach for visualizing localization of specific protein expression within tissue sections using antibodies. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization christina n.
Zebrafish whole mount immunohistochemistry youtube. Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. The researcher should optimize the precise conditions for a particular assay. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. Useful tip for better zebrafish wholemount ihc staining. For icc experiments, cells must be attached to a microscope slide. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. In whole mount ihc, the distribution of an antigen can be mapped rapidly and reliably in the embryos. Since the optimization of the multicolor immunostaining protocol is. Sequential immunofluorescence and immunohistochemistry. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. The principles of ihc have been known since the 1930s, but it was not until 1942 that the first ihc study was reported. Zebrafish whole mount immunohistochemistry reagents.
In this chapter, i will describe the basic technique developed for wholemount labeling of. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. Dec 20, 2007 the in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Highresolution in situ hybridization to wholemount. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. Here, we outline our protocol for embedding, serial sectioning, staining, and visualizing zebrafish embryos embedded in jb4 plastic resin a glycol methacrylatebased medium that results in. Incubate for 10 min utes at room temperature in methanolperoxidase. Trouble shooting in immunohistochemistry the enzymatic activation of the chromogen results in a visible reaction product at the antigen site.
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. With any luck, you will need to collect only one ribbon containing gills one bisected half and spinal cord the other bisected half, along with a nice sampling of internal organs. Ihc detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence. 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. Staining slides for a discussion of antigfp and secondary antibodies. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Prepare formalinfixed, paraffinembedded tissue sections step 18. Oct 28, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. 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 immunohistochemical and immunofluorescence.
Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Immunohistochemistry staining for frozen tissues acetonefixed ebioscience immunohistochemistry on schneider line 2 cells. Representative imaging of wholemount immunohistochemistry of zebrafish danio rerio embryo 48. Green fluorescent protein labeling of dopaminergic neurons. We currently offer a continuously expanding product list for research in zebrafish. All zebra fish were maintained and bred in similar culture conditions by following standard guidelines for fish care and maintenance protocols 18. The zebrafish, danio rerio, has become a widely used organism in the study of developmental biology. Rnadna fish on metaphases rnadna fish dna step only nick translation protocol for direct probe labeling edith slide preparation by. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is.
Zebrafish whole mount highresolution double fluorescent in. Pdf wholemount immunohistochemistry for antif59 in. Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific. Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues. 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. In wholemount ihc, the distribution of an antigen can be. Zebrafish were originally used for environmental research to gain information on the hazards of potential water pollutants, as they were easily obtainable, inexpensive, and easy to maintain 1. Developmental stages were determined according to 19. Cryopreservation and in vitro fertilization ivf protocols.
Zebrafish sample preparation for whole mount immunohistochemistry. This protocol describes ish of digoxigeninlabeled antisense rna. This gives a clearer idea of where the target protein of interest is expressed within the. 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. Therefore, sectioning is often critical to examine cellular localization of proteins. Commercially available antibodies will have a data sheet that will list the recommended. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Embedding, serial sectioning and staining of zebrafish.
Since then, major improvements have been made in tissue fixation and sectioning methods, antigenepitope retrieval, antibody conjugation, immunostaining methods and. 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. The antibodyantigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye. Rna fish following immunofluorescence when if and fish are to be combined, we prefer to perform if under rnasefree conditions prior to the fish, as the formamide treatment during the fish procedure is sometimes incompatible with preservation of. One for alla highly efficient and versatile method for fluorescent. Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Mar 12, 2020 protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. Staining protocol the standard protocol is intended for use as a guideline in developing antibodyspecific procedures. Ihc immunohistochemistry protocol enquire bioreagents. Immunohistochemistry ihc is a powerful research tool to localize specific antigens in whole mount or tissue sections of embryos with labeled antibodies based on antigenantibody interactions. Therefore, it is important to know the characteristics of the antibody.
Xenopus wholemount immunohistochemistry protocol general information. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. In order to create a new protocol or add a comment, you must have a wiki account. The in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Immunofluorescence protocol for confocal microscopy. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna. Antibody list tested in zebrafish and medaka with or without the. Immunohistochemistry ihc protocolparaffin section protocol. Full text pdf manual immunohistochemistry staining of mouse tissues using the avidinbiotin complex abc technique robert d.
However, do not rock at high speed since the embryos are somewhat fragile. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. Because the fish has been cut to one side of the spinal cord, you will be cutting toward the midline on one half of the fish, and away from the midline on the other. Histology and immunohistochemistry online protocols. An example of a section through an immunostained embryo in whole mount is pictured in figure 4d. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Davis introduction wholemount immunohistochemistry is the localization of antigens in unsectioned tissues using specific antibodies. Here we describe an ihc protocol for localizing the myosin heavy. Immunohistochemistry protocol for frozen tissue download a pdf. As in figure 3a, we performed multicolor whole mount fluorescent immunostaining on zebrafish and. The protocol is a modified version of the standard in situ. Whole mount immunohistochemistry and in situ hybridization of.
To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Zebrafish whole mount highresolution double fluorescent. Immunohistochemistry ihc is a technique commonly used for morphological characterization of tumors or other tissue malignancies. General wholemount immunohistochemistry of zebrafish danio. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. Basics and methods, a new book that aims to make the technique accessible to a wide audience. The zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf. Immunofluorescence and fluorescent in situ hybridization. Betagal staining whole mount karl clark betagal staining allows identification of embryonic tissuescells expressing lacz marker protein by development of pigmented blue product in the presence of lacz enzymatic activity. Overview of immunohistochemistry thermo fisher scientific za.
The sections were incubated with a biotinylated anticytokeratin 18 antibody and then detected using a thermo fisher streptavidindylight 633 conjugate cat. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence. Whole mount immunohistochemistry and in situ hybridization. Wash slides in 100% alcohol twice for two minutes each. Pdf wholemount immunohistochemistry for antif59 in zebrafish. 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. Whole mount immunohistochemistry in zebrafish embryos and. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. Proteins 502 31 wholemount immunohistochemistry by claytus a. The following immunohistochemistry protocol will discuss immunohistochemistry in formalinfixed, paraffinembedded tissues. 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. Histological techniques are critical for observing tissue and cellular morphology.