Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides. Sequential immunofluorescence and immunohistochemistry. Etiology and functional validation of gastrointestinal. The antibodyantigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye. The following immunohistochemistry protocol will discuss immunohistochemistry in formalinfixed, paraffinembedded tissues. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. Here we present a novel method to efficiently retrieve the antigen in. Antibody list tested in zebrafish and medaka with or without the.
Wash embryo with pbs twice for 1020 minutes each at rt. Immunohistochemistry ihc is a common approach for visualizing localization of specific protein expression within tissue sections using antibodies. Histological methods protocols zfin community wiki. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. Immunofluorescence and fluorescent in situ hybridization. The zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. This sequential ifihc protocol for cryosectioned zebrafish will provide. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is. 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. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna.
Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence. Not only are anatomical structures in zebrafish small, cells are also smaller compared to. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization christina n. Wash slides in 100% alcohol twice for two minutes each. 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. Proteins 502 31 wholemount immunohistochemistry by claytus a. Staining protocol the standard protocol is intended for use as a guideline in developing antibodyspecific procedures. Trouble shooting in immunohistochemistry the enzymatic activation of the chromogen results in a visible reaction product at the antigen site.
Whole mount immunohistochemistry in zebrafish embryos and. 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. Please optimize the procedure since experimental conditions can vary for different tissue samples. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf chapter pdf available january 2016 with 178 reads how we measure reads. Wildtype zebra fish tu strain were obtained from zirc university of oregon, eugene, usa. Since then, major improvements have been made in tissue fixation and sectioning methods, antigenepitope retrieval, antibody conjugation, immunostaining methods and.
Green fluorescent protein labeling of dopaminergic neurons. Zebrafish whole mount immunohistochemistry youtube. 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. Histological techniques are critical for observing tissue and cellular morphology. Davis introduction wholemount immunohistochemistry is the localization of antigens in unsectioned tissues using specific antibodies. Zebrafish sample preparation for whole mount immunohistochemistry. 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. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna sequences on chromosomes. Overview of immunohistochemistry thermo fisher scientific za. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. General wholemount immunohistochemistry of zebrafish danio.
The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Full text pdf manual immunohistochemistry staining of mouse tissues using the avidinbiotin complex abc technique robert d. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. In wholemount ihc, the distribution of an antigen can be. Immunohistochemistry protocol for frozen tissue download a pdf. Mar 12, 2020 protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. 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. 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.
Dec 20, 2007 the in situ hybridization ish technique allows the sites of expression of particular genes to be detected. In situ hybridization ish nucleic acid retrieval principle, technique and. General wholemount immunohistochemistry of zebrafish. Wholemount in situ hybridization wish is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. Zebrafish whole mount immunohistochemistry reagents. Developmental stages were determined according to 19. The principles of ihc have been known since the 1930s, but it was not until 1942 that the first ihc study was reported.
Immunofluorescence protocol for confocal microscopy. Cryopreservation and in vitro fertilization ivf protocols. Histology and immunohistochemistry online protocols. The zebrafish, danio rerio, has become a widely used organism in the study of developmental biology. In order to create a new protocol or add a comment, you must have a wiki account.
However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. 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. Wholemount immunohistochemical and immunofluorescence. Although many immunohistochemistry protocols have been optimized for. Jb4 resin can also be used to analyze gfp signals in sections from both mosaically expressing figure 4e and transgenic figure 4f fish.
However, do not rock at high speed since the embryos are somewhat fragile. This protocol describes ish of digoxigeninlabeled antisense rna probes to whole mount. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Rnadna fish on metaphases rnadna fish dna step only nick translation protocol for direct probe labeling edith slide preparation by. Immunohistochemistry ihc uses antibodies to detect the location of proteins and other antigens in tissue sections. The sections were incubated with a biotinylated anticytokeratin 18 antibody and then detected using a thermo fisher streptavidindylight 633 conjugate cat.
Xenopus wholemount immunohistochemistry protocol general information. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization. This gives a clearer idea of where the target protein of interest is expressed within the. Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific.
Zebrafish whole mount highresolution double fluorescent in. In situ hybridization ish nucleic acid retrieval principle, technique and protocol. 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. In addition, it is important to determine if the antibody will work with the sample that you wish to study.
Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. 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. Here we describe an ihc protocol for localizing the myosin heavy. Rna fish on whole mount blastocyst rna fish on whole mount 6.
Immunohistochemistry ihc protocolparaffin section protocol. Prepare formalinfixed, paraffinembedded tissue sections step 18. Commercially available antibodies will have a data sheet that will list the recommended. Zebrafish are a tropical, freshwater vertebrate species belonging to the family cyprinidae.
Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf. The researcher should optimize the precise conditions for a particular assay. Immunohistochemistry ihc is a technique commonly used for morphological characterization of tumors or other tissue malignancies. Useful tip for better zebrafish wholemount ihc staining.
In this chapter, i will describe the basic technique developed for wholemount labeling of. This guide highlights critical steps in the ihc protocol and demonstrates how protocol changes can affect the final. 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. Highresolution in situ hybridization to wholemount. For icc experiments, cells must be attached to a microscope slide. Oct 28, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. This is an official page of the university of massachusetts medical school. Ihc detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence. 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. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. 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. Flat mount preparation for observation and analysis of. 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.
Ihc immunohistochemistry protocol enquire bioreagents. Therefore, it is important to know the characteristics of the antibody. 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. 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. 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.
Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. 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. Basics and methods, a new book that aims to make the technique accessible to a wide audience. 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. Embedding, serial sectioning and staining of zebrafish. Representative imaging of wholemount immunohistochemistry of zebrafish danio rerio embryo 48. Staining slides for a discussion of antigfp and secondary antibodies. Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. Pdf wholemount immunohistochemistry for antif59 in zebrafish. Whole mount immunohistochemistry and in situ hybridization.
For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Mount the coverslips on a slide in glycerolbased mounting medium. Whole mount immunohistochemistry and in situ hybridization of. Therefore, sectioning is often critical to examine cellular localization of proteins. Incubate for 10 min utes at room temperature in methanolperoxidase.
In whole mount ihc, the distribution of an antigen can be mapped rapidly and reliably in the embryos. Deparaffinize slides in two changes of xylene for five minutes each. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. One for alla highly efficient and versatile method for fluorescent. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. The in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Different antibodies and tissues may require changes to this procedure. Immunohistochemistry staining for frozen tissues acetonefixed ebioscience immunohistochemistry on schneider line 2 cells. Nuclei stained with propidium iodide are colored blue.
This protocol describes ish of digoxigeninlabeled antisense rna. Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. 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. Pdf wholemount immunohistochemistry for antif59 in. Since the optimization of the multicolor immunostaining protocol is.