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. One for alla highly efficient and versatile method for fluorescent. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. Therefore, it is important to know the characteristics of the antibody. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Incubate for 10 min utes at room temperature in methanolperoxidase. The zebrafish, danio rerio, has become a widely used organism in the study of developmental biology.
Immunohistochemistry staining for frozen tissues acetonefixed ebioscience immunohistochemistry on schneider line 2 cells. Therefore, sectioning is often critical to examine cellular localization of proteins. Whole mount immunohistochemistry in zebrafish embryos. The antibodyantigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye. The researcher should optimize the precise conditions for a particular assay. Sequential immunofluorescence and immunohistochemistry. 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.
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. The in situ hybridization ish technique allows the sites of expression of particular genes to be detected. Here we present a novel method to efficiently retrieve the antigen in. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna. 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. Zebrafish whole mount highresolution double fluorescent in. Embedding, serial sectioning and staining of zebrafish. For icc experiments, cells must be attached to a microscope slide. Wholemount in situ hybridization wish is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. Davis introduction wholemount immunohistochemistry is the localization of antigens in unsectioned tissues using specific antibodies. Prepare formalinfixed, paraffinembedded tissue sections step 18.
Wash embryo with pbs twice for 1020 minutes each at rt. Zebrafish sample preparation for whole mount immunohistochemistry. In whole mount ihc, the distribution of an antigen can be mapped rapidly and reliably in the embryos. 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. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is. Proteins 502 31 wholemount immunohistochemistry by claytus a. This guide highlights critical steps in the ihc protocol and demonstrates how protocol changes can affect the final. Oct 28, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific.
Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides. Since then, major improvements have been made in tissue fixation and sectioning methods, antigenepitope retrieval, antibody conjugation, immunostaining methods and. This protocol describes ish of digoxigeninlabeled antisense rna. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. 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. Deparaffinize slides in two changes of xylene for five minutes each. This protocol describes ish of digoxigeninlabeled antisense rna probes to whole mount. Rnadna fish on metaphases rnadna fish dna step only nick translation protocol for direct probe labeling edith slide preparation by. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization christina n. Green fluorescent protein labeling of dopaminergic neurons. The zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. General wholemount immunohistochemistry of zebrafish. Inadequately fixation can make tissues dehydrated during tissue processing and become hard and brittle. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development.
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. Immunohistochemistry ihc is a common approach for visualizing localization of specific protein expression within tissue sections using antibodies. Pdf wholemount immunohistochemistry for antif59 in. It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna sequences on chromosomes. Trouble shooting in immunohistochemistry the enzymatic activation of the chromogen results in a visible reaction product at the antigen site. However, do not rock at high speed since the embryos are somewhat fragile. Although many immunohistochemistry protocols have been optimized for. Rinse the tissue with running tap water for 30min40min to eliminate the formaldehyde. This gives a clearer idea of where the target protein of interest is expressed within the. Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. General wholemount immunohistochemistry of zebrafish danio.
Whole mount immunohistochemistry and in situ hybridization. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. This is an official page of the university of massachusetts medical school. 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. Cryopreservation and in vitro fertilization ivf protocols. Mount the coverslips on a slide in glycerolbased mounting medium. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Developmental stages were determined according to 19. Whole mount immunohistochemistry and in situ hybridization of larval and adult zebrafish dental tissues. Here we describe an ihc protocol for localizing the myosin heavy.
Since the optimization of the multicolor immunostaining protocol is. Immunohistochemistry protocol for frozen tissue download a pdf. Immunohistochemistry ihc uses antibodies to detect the location of proteins and other antigens in tissue sections. Immunohistochemistry is a method of detecting the presence of specific proteins in cells or tissues and consists of the following steps. Ihcicc protocol guide 4 tissue and cell samples must be appropriately harvested and prepared for each ihcicc study. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. The sections were incubated with a biotinylated anticytokeratin 18 antibody and then detected using a thermo fisher streptavidindylight 633 conjugate cat. 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. Staining protocol the standard protocol is intended for use as a guideline in developing antibodyspecific procedures. Whole mount immunohistochemistry and in situ hybridization of. Immunohistochemistry ihc is a technique commonly used for morphological characterization of tumors or other tissue malignancies. Full text pdf manual immunohistochemistry staining of mouse tissues using the avidinbiotin complex abc technique robert d. Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf chapter pdf available january 2016 with 178 reads how we measure reads.
Jb4 resin can also be used to analyze gfp signals in sections from both mosaically expressing figure 4e and transgenic figure 4f fish. The principles of ihc have been known since the 1930s, but it was not until 1942 that the first ihc study was reported. In addition, it is important to determine if the antibody will work with the sample that you wish to study. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19. Zebrafish whole mount highresolution double fluorescent. 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. Ihc detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence. In wholemount ihc, the distribution of an antigen can be. Commercially available antibodies will have a data sheet that will list the recommended. Fix dissected tissues with 10% formalin for no less than 48 hours at room temperature. The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. As in figure 3a, we performed multicolor whole mount fluorescent immunostaining on zebrafish and. Histological techniques are critical for observing tissue and cellular morphology. Wildtype zebra fish tu strain were obtained from zirc university of oregon, eugene, usa.
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 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 are a tropical, freshwater vertebrate species belonging to the family cyprinidae. Rna fish on whole mount blastocyst rna fish on whole mount 6.
Wholemount immunohistochemical and immunofluorescence. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence. Useful tip for better zebrafish wholemount ihc staining. In situ hybridization ish nucleic acid retrieval principle, technique and. Ihc immunohistochemistry protocol enquire bioreagents. Flat mount preparation for observation and analysis of. Representative imaging of wholemount immunohistochemistry of zebrafish danio rerio embryo 48. This sequential ifihc protocol for cryosectioned zebrafish will provide. Antibody list tested in zebrafish and medaka with or without the. 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. 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.
Histology and immunohistochemistry online protocols. Immunohistochemistry ihc protocolparaffin section protocol. Staining slides for a discussion of antigfp and secondary antibodies. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. In situ hybridization ish nucleic acid retrieval principle, technique and protocol.
The protocol is a modified version of the standard in situ. Whole mount immunohistochemistry in zebrafish embryos and. Etiology and functional validation of gastrointestinal. Zebrafish whole mount immunohistochemistry reagents.
Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. 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 methods protocols zfin community wiki. Overview of immunohistochemistry thermo fisher scientific za. Not only are anatomical structures in zebrafish small, cells are also smaller compared to. Zebrafish whole mount immunohistochemistry youtube. Highresolution in situ hybridization to wholemount. Mar 12, 2020 protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization.
Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. Dec 20, 2007 the in situ hybridization ish technique allows the sites of expression of particular genes to be detected. The following immunohistochemistry protocol will discuss immunohistochemistry in formalinfixed, paraffinembedded tissues. In order to create a new protocol or add a comment, you must have a wiki account. Immunofluorescence and fluorescent in situ hybridization. Different antibodies and tissues may require changes to this procedure. 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. 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. 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. Wash slides in 100% alcohol twice for two minutes each. We currently offer a continuously expanding product list for research in zebrafish. Basics and methods, a new book that aims to make the technique accessible to a wide audience.
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