The researcher should optimize the precise conditions for a particular assay. Images are maximum projections of confocal series, contrast enhanced in adobe photoshop. Antibody staining protocols wholemount staining of biotindextran injected zebrafish. Twocolor fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. Since then, major improvements have been made in tissue fixation and sectioning methods, antigenepitope retrieval, antibody conjugation, immunostaining methods and. Individual fish counting was done at 3 dpf, 30 dpf, 60 dpf and table 1. Transfer to millonigs buffer without glutaraldehyde. Zebrafish whole mount immunohistochemistry procedure based on protocol and information kindly provided by. The zebrafish, danio rerio, has become a widely used organism in the study of developmental biology. Researchers can generate highresolution images for protein localization studies and now have the ability to quantitate the fluorescence signals using sophisticated imaging software. Request pdf general wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol immunohistochemistry ihc is a powerful research tool to localize specific.
The principles of ihc have been known since the 1930s, but it was not until 1942 that the first ihc study was reported. The antibodyantigen interaction is visualized using either chromogenic detection with a colored enzyme substrate, or fluorescent detection with a fluorescent dye. Whole mount immunohistochemistry and in situ hybridization. Immunofluorescence method for ihc detection thermo fisher. Zebrafish are a tropical, freshwater vertebrate species belonging to the family cyprinidae. Histology and immunohistochemistry online protocols.
Representative imaging of wholemount immunohistochemistry of zebrafish. Sectioning tissue sections 57 m thick are cut from paraffinembedded blocks on a microtome and mounted from warm water 40c onto adhesive microscope slides. Feel free to add new protocols to the appropriate section or add comments to any existing protocol. Nick translation protocol for direct probe labeling edith.
Wash slides in 100% alcohol twice for two minutes each. Commercially available antibodies will have a data sheet that will list the recommended. However, do not rock at high speed since the embryos are somewhat fragile. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. Nonetheless, nonspecific binding in thick tissues remains a major problem. For tissue stored at 80c, remove from freezer and equilibrate at 20c for about 15 minutes before sectioning. Immunohistochemistry method deparaffinize sections and rehydrate using pbs. Ihc detection of cytokeratin 18 in human colon carcinoma tissue by immunofluorescence. Rnadna fish on metaphases rnadna fish dna step only nick translation protocol for direct probe labeling edith slide preparation by. Education guide immunohistochemical staining methods fifth. Immunofluorescence method for ihc detection thermo. Immunofluorescence protocol if protocol immunohistochemistry. Therefore, it is important to know the characteristics of the antibody. When working with cells this technique is generally referred to as immunocytochemistry.
Zebrafish sample preparation for whole mount immunohistochemistry. A rapid and nondestructive protocol for wholemount bone staining of small fish and xenopus article pdf available in scientific reports 81 december 2018 with 365 reads how we measure reads. Place embryo in a 5 ml bijous in fixative for 1 hour. 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.
Flat mount preparation for observation and analysis of. In situ hybridization and immunohistochemistry are wellestablished and frequently used techniques to study the presence of mrna and protein. Immunofluorescence protocol for confocal microscopy. The protocol is a modified version of the standard in situ hybridization. The 5th edition is available in print and within the zfin protocol wiki. Achieving reliability and reproducibility of immunostains. Dehydrate with 100% meoh twice 10 min each and mount in a 2.
In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish. Samples are immediately fixed in carnoys solution and left overnight. General wholemount immunohistochemistry of zebrafish danio. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. Nonetheless, nonspecific binding in thick tissues remains a major problem of whole mount immunohistochemistry 1,1017. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. A novel protocol of whole mount electroimmunofluorescence. Please optimize the procedure since experimental conditions can vary for different tissue samples. Immunohistochemistry protocol for frozen tissue download a pdf. Zebrafish whole mount highresolution double fluorescent. 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. 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 immunohistochemistry in zebrafish embryos.
Zebrafish whole mount immunohistochemistry reagents. Histology and immunohistochemistry protocols from other online sources. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. Useful tip for better zebrafish wholemount ihc staining. To get better penetration of the staining reagents into tissues, incubation times must often be extended for a couple days in conjunction with the use of nonionic detergent, e. Frequently asked questions continues to provide a comprehensive and yet concise stateoftheart overview of the major issues specific to the field of immunohistochemistry. A guide for the laboratory use of zebrafish danio rerio. For transmission electron microscopy of fish tissues, we generally prepare 4% glutaraldehyde in millonigs buffer. Davis introduction wholemount immunohistochemistry is the localization of antigens in unsectioned tissues using specific antibodies. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. This mixture has the same refractive index as yolk and clears the embryos very well.
The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. 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. For a slightly less rigid mounting medium, use a lower concentration of agarose 0. Jul 24, 2018 the mission of the national institute of environmental health sciences is to discover how the environment affects people in order to promote healthier lives. The antibody is conjugated to either a fluorescent or enzymatic label, and the location of the label seen through a. Feeding protocol for each feeding regimen and feed group after 6 dpf i. Whole mount immunohistochemistry in zebrafish embryos and. A guide for the laboratory use of zebrafish danio brachydanio rerio. Fix for 1 hour, wash in pbs 1% triton then permeabilize the egg membrane in in ice cold acetone pbs for 8 minutes only. Basics and methods, a new book that aims to make the technique accessible to a wide audience. Xenopus wholemount immunohistochemistry protocol general information. This application looks and works like any windows help. Fluorescence detection is emerging as a popular alternative to traditional chromogenbased ihc.
Zebrafish whole mount immunohistochemistry youtube. Deparaffinize slides in two changes of xylene for five minutes each. Manual immunohistochemistry staining of mouse tissues using the avidinbiotin complex abc technique robert d. Analysis of apoptosis in zebrafish embryos by wholemount. Methyl cellulose mounting protocols zfin community wiki. Pretreat antigen retrieval the sample with one of the following methods. Immunohistochemistry is a laboratory technique utilized for the visual detection of antigens in tissue. 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. 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. Frozen section technique a method for preparation of frozen sections pathology innovations.
Wholemount immunohistochemistry for antif59 in zebrafish embryos 15 days post fertilization dpf. Whole mount immunohistochemistry for the glun1 subunit of the nmdatype glutamate receptor reveals expression of glutamate receptor subunits throughout developing zebrafish muscle at 23 hpf figure 2. Protocols are organized into sections corresponding to the chapters of the zebrafish book, 5th edition 4th edition online. 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. Proteins 502 31 wholemount immunohistochemistry by claytus a.
Immunohistochemistry ihc is a common approach for visualizing localization of specific protein expression within tissue sections using antibodies. Wholemount immunohistochemistry of the brain request pdf. Dec 20, 20 whole mount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Different feeds and feeding regimens have an impact on. Ihc staining protocol for whole mount samples abcam. To develop a new method of whole mount immunostaining that improves the penetration of staining reagents into the cornea and decreases nonspecific binding and background. This guide highlights critical steps in the ihc protocol and demonstrates how protocol changes can affect the final.
Adult mouse corneas were fixed overnight in 4% paraformaldehyde or a mixture of 4% paraformaldehyde and 0. Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. Whole mount staining of zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. Wash embryo with pbs twice for 1020 minutes each at rt. Immunohistochemistry ihc guide and troubleshooting md.
Oct 28, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. The following day, the tissues are embedded into paraffin. The mission of the national institute of environmental health sciences is to discover how the environment affects people in order to promote healthier lives. Introduction immunohistochemistry is used to identify the location and distribution of target antigens in cells or tissues by staining with a specific antibody. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. In this chapter, a general protocol for wholemount immunofluorescent labeling of zebrafish embryos and larvae is described. Tissue samples are dissected from animals follow an appropriate protocol.
Overview of immunohistochemistry thermo fisher scientific ar. Methyl cellulose is especially good for orienting young embryos less than 10 hpf old. Staining slides for a discussion of antigfp and secondary antibodies. This is an official page of the university of massachusetts medical school. These techniques are widely used by histologypathology laboratories and life science researchers as a method to detect abnormal cells or to characterize. Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain.
This corresponds approximately with the timing of motor neuron innervation. This is where zebrafish researchers can share experimental protocols and tips with the rest of the research community. 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. Although many immunohistochemistry protocols have been. Immunohistochemistry protocols national institute of. The following immunohistochemistry protocol will discuss immunohistochemistry in formalinfixed, paraffinembedded tissues. Not only are anatomical structures in zebrafish small, cells are also smaller. Fixative 4% formaldehyde in pbs freshly prepared 3.
Here, we present a protocol for fluorescent antibodymediated. The zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. The fixative should be chosen carefully, depending on what has been previously used successfully with the antibody in cryosections, and also on the target protein. Pdf a rapid and nondestructive protocol for wholemount. Pdf wholemount immunohistochemistry for antif59 in zebrafish. Cells grown on cover slips or on commercially available incubation chambers. 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. Education guide immunohistochemical staining methods. General wholemount immunohistochemistry of zebrafish. 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.
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. 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. Mount tissue in permount between two coverslips, using strips of teflon tape or pieces of. A 28h wholemount zebrafish embryo labeled with an antibody which recognizes the transcription factor, pax6 brown label showing a lateral view with anterior to the left.
We recommend following the zebra fish whole mount staining procedure provided. Immunohistochemistry standard operating protocol edrn. Rna fish on whole mount blastocyst rna fish on whole mount 6. Whole mount immunohistochemistry and in situ hybridization of. Fluorescence in situ hybridization fish on human chromosomes and interphase nuclei. The sections were incubated with a biotinylated anticytokeratin 18 antibody and then detected using a thermo fisher streptavidindylight 633 conjugate cat. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. We currently offer a continuously expanding product list for research in zebrafish. B the 3d larvae labeled with antiacetylated tubulin antibody brown, lateral view with anterior to the left.
The whole mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. Immunohistochemistry on whole mount embryos paul mcdonald immunofluorescence. Here we provide a protocol for usual immunohistochemistry. Immunohistochemistry ihc is a technique commonly used for morphological characterization of tumors or other tissue malignancies.
Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Immunohistochemistry and molecular morphology shanrong shi, jiang gu, clive roy taylor eaton pub. Ihc immunohistochemistry protocol enquire bioreagents. Fix small pieces of tissue at room temperature for 2 h, then hold overnight at 4 0 c. With links to the authors immunohistochemical laboratory website, this.