Whole mount dapi staining does anybody have experiencegood advice on using the dapi stain on fresh tissue thickness between 0. Browse our extensive catalog for polyclonal, monoclonal, and conjugated antigfp antibodies. I already used ha antibody from roche, 3f10 anti rat monoclone. It especially enables the improvement of the staining reagent penetration into the tissues by electric force. Zebrafish whole mount immunohistochemistry protocol abcam. Gb mount is a waterbased mounting media for using with frozen section, immunoperoxidase and immunofluorescent procedures. Bgal staining whole mount college of biological sciences.
Clearing and staining for larval fish cartilage and bone. Dechorionate 26 hr embryos pharyngula stage carefully with two fine. A novel protocol of whole mount electroimmunofluorescence staining. Automated deepphenotyping of the vertebrate brain elife. Mbp antibody staining protocol for immunohistochemistry. Aec, apred, and apblue are soluble in alcohol and organic solvents. In the present study, we propose and demonstrate a new protocol for whole mount bone staining in small fish medaka and zebrafish and xenopus frogs. Staining of 30 h fish or older is improved by adding triton x100 to a final concentration of 0. Zebrafish whole mount immunohistochemistry youtube. Introducing rabbit mab to community antibodies zfin. This article is from journal of visualized experiments. Flat mount imaging of mouse skin and its application to the. Ventral view with some lateral tilt, anterior to bottom. Thorough washing seems to be the most important factor in reducing background and should not be minimized.
Neurotoxicity assessment using zebrafish sciencedirect. A direct fluorescent monoclonal antibody staining method has been developed for rapid detection of viable cells of vibrio cholerae o1 and o9. Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development will need to be much longer than normal. This antibody is crossadsorbed against bovine igg, goat igg, rabbit igg, rat igg, human igg and human serum.
Immunohistochemistry of wholemount and sectioned zebrafish tissue. Wholemount in situ hybridization analyses at 24 hpf using scltal1 riboprobe showed staining within the bilateral cranial cells and in the intermediate cell mass icm. 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. Method for whole mount antibody staining in chick protocol. Ihc wholemount antivasavas antibody ab209710 immunohistochemical analysis whole mount of paraformaldehydefixed zebrafish embryo labeling. Wholemount immunohistochemistry ihc is a valuable tool for investigation of protein expression during zebrafish embryonic development.
Visualization of both tunel staining and antiactive caspase3 staining section 2. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Intrafollicular thyroid hormone staining in wholemount. A rapid and nondestructive protocol for wholemount bone. Wholemount immunostaining methods to study the blood and. The whole mount electrostaining readily recognized filopodia and lamellipodia at the leading edge of epithelium debridement by clsm. High resolution whole mount in situ hybridization within. The use of znp1 and f6 coupled with the secondary antibody.
Myelin basic protein mbp is a major constituent of the myelin sheath of oligodendrocytes and schwann cells in the central nervous system and the peripheral nervous system, respectively. Automated deepphenotyping of entire brains in zebrafish mutants. Nuclei stained with propidium iodide are colored blue. If you are by chance staining the vasculature, also make sure that specimen are sufficiently prehybidized to inactivate endogenous. Cells should be rinsed with pbs to remove serum proteins prior to antibody staining. All incubations should be performed on ice and with minimal light exposure. The following procedure describes the procedure for whole mount staining of chick or mouse embryos. The staining for shh was observed in the notochord. Use clean glassware preferably sterile and fresh reagents.
Antibody staining protocol cells should be suspended at approximately 110x106 cellsml. For embryos at early developement stage up to 18h, dehydrate in 100% methanol, clear for a few minutes in methylsalycilate, and mount in permount. Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. It is most abundant in hemopoietic system and contains seven exons distributed over 32. Nov 29, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. 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. We present three newly developed methods for preparing whole mounts of mammary glands from geneticallyengineered mice. In recent years, the zebrafish embryo has emerged as a popular model. Wholemount immunofluorescence university of louisville. 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. Lab recipes antibody staining protocol pdf instructions for making bridged coverslips pdf. As in figure 3a, we performed multicolor whole mount fluorescent immunostaining on zebrafish and medaka embryos with the same antibodies. I have sv2 antibody from developmental studies hybridoma bank dshb.
This material is from the 4th edition of the zebrafish book. Tissue slides processed by these methods should be coverslipped with gb mount. Oct 28, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Antimyosin heavy chain mouse monoclonal f59 primary antibody was used at a concentration of 1. Initial formation of zebrafish brain ventricles occurs independently of. Zebrafish sample preparation for whole mount immunohistochemistry. The zebrafish neuromuscular junction, like other vertebrates, is mediated by acetylcholine acting at nicotinic acetylcholine receptors. Whole mount antibody staining of zebrafish embryos for. As the incubations in whole mount staining are very long, microbial contamination can become a problem. B the 3d larvae labeled with antiacetylated tubulin antibody brown, lateral view with anterior to the left.
It is recommended that as incubations can be very long in whole mount staining, the antibody should be diluted in blocking buffer containing 0. Wholemount staining of biotindextran injected embryos. Wholemount in situ hybridization is a powerful technique that enables scientists to understand the molecular basis of embryonic development. This can be accomplished either by whole mount in situ hybridization wish using riboprobes or by whole mount immunohistochemistry using antibodies. Analysis of apoptosis in zebrafish embryos by wholemount.
For observation using a dissecting microscope, mount embryos directly in stop solution and methylcellulose. Whole mount staining of biotindextran injected embryos. During normal development until 10 days postfertilization dpf, the number of. Here, we present a protocol for fluorescent antibodymediated detection of proteins in whole preparations of zebrafish embryos and. General wholemount immunohistochemistry of zebrafish. Rna in situ protocol pdf whole mount antibody staining. Since the heating method had been successfully applied to whole mount preparations prior to sectioning and antibody staining on sections, we tested the method on whole mount immunostainings. Can anyone give a procedure for whole mount sv2 staining. Can anyone give a procedure for whole mount sv2 staining for zebrafish larvae 120hpf.
Figure 1f,g,ik, cytochemistry figures 1h,2f,g, immunohistochemistry, or more simply using. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. 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. In addition, we can deliver the gene sequences of rmabs to enhance reproducibility and ease antibody engineering for special. Can anyone give a procedure for whole mount sv2 staining for. Double in situ hybridization techniques in zebrafish. For observation using a compound microscope, mount embryos in 100% glycerol. Photoconversion of fluorescently labeled profiles for em. Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development will need to be much longer than normal immunocytochemistry. 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. In brief, fixedembryos after rehydration were heated in 150 mm trishcl at ph 9. Ihc staining protocol for whole mount samples abcam. This protocol is essentially used for the spatial and temporal characterization of novel antibodies in chick 2,3, as well as for the use of known antigenic markers to determine embryonic malformations following insult 4.
Zebrafish whole mount highresolution double fluorescent. The whole mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. In situ hybridization and immunohistochemistry are wellestablished and frequently used techniques to study the presence of mrna and protein. Whole mount indicates that the entire embryo will be used, not just a tissue slice.
A simple wholemount staining protocol for bone and or cartilage in adults and larvae. In whole mount ihc, the distribution of an antigen can be mapped rapidly and reliably in the embryos. Use of phospholipase a2 for antigen retrieval in zebrafish. Whole mount staining of biotindextran injected zebrafish embryos.
Whole mount in situ hybridization solutions chapter 10 recipes abc solution avidinbiotin hrp complex. Immuno histo chemistry on whole mount zebrafish embryos. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. Recommendations for sample handling times and volumes given are a general. Western blot, flow cytometry, immunohistochemistry, blockade of receptorligand interaction, cytofready. Tested in immunofluorescence if, immunocytochemistry icc, immunohistochemistry ihc and flow cytometry flow applications.
Whole mount staining of zebrafish embryos, now commonly used, requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilized. Covering animal models such as the fruit fly, zebra fish, chicken, and mouse, this detailed book views in situ hybridization, reporter gene expression, and immunohistochemical staining methods, as well as rna interference, morpholino, or transgenic techniques through the prism of these models. Feb 02, 2009 this video demonstrates the different steps in performing whole mount antibody staining in young chick embryos. Additionally data quantification of lymphaticblood vasculature is often most accurate with whole mount immunostaining. Kao 1, 2 1 department of ophthalmology, university of cincinnati, cincinnati, oh. The mouse notch3 antibody has been validated for the following applications. Nov 24, 2009 whole mount preparations of mouse mammary glands are useful for evaluating overall changes in growth and morphology, and are essential for detecting and evaluating focal or regionallylocalized phenotypes that would be difficult to detect or analyze using other techniques. The fully updated volume examines cuttingedge techniques on a representative range of animals, including widely used genetic model systems, such as the fruit fly, zebra fish, chicken, and mouse, as well as noncanonical experimental systems opened up through the advent of genome editing.
Staining wholemount embryos with pap protocols zfin. Here, we present a highresolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining. Validated research applications for gfp antibodies include elisa, electron microscopy, flow cytometry, cytofready, icc, immunofluorescence, ihc, ihcfrozen, ihcparaffin, ihc whole mount, ip, simple western, and western blot. Wholemount immunostaining was carried out using antiphosphohistone h3. Whole mount immunohistochemical staining of zebrafish embryos was carried out as described previously incardona et al. Immunohistochemistry ihc is the most common application of immunostaining.
Immunohistochemistry ihc is a powerful research tool to localize specific antigens in whole mount or tissue sections of embryos with labeled antibodies based on antigen antibody interactions. If the antibody yields a good signal to noise ratio, the antibody is then compatible with the methanol treatment and should work well in whole mount. Ihc takes its name from the roots immuno, in reference to antibodies used in the procedure, and histo, meaning tissue. Immunohistochemistry is also an important complement to gene expression, as translation and transcription may not be concurrent. Bgal staining allows identification of embryonic tissuescells expressing lacz marker protein by development of pigmented blue product in the presence of lacz enzymatic activity. This video demonstrates the different steps in performing whole mount antibody staining in young chick embryos. And finally, hybridization refers to the complementary binding of. A novel protocol of whole mount electroimmunofluorescence. The wholemount casp3 assay is optimized for analysis of fixed embryos between the 4cell stage and 32 hrpostfertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wildtype mrnas, and exposure to chemicals. To assess pharmacological effects of drugs on the optic nerves, motor neurons, and dopaminergic neurons, we performed whole mount immunostaining and visualized different neuronal.
Whole mount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Immuno histo chemistry on whole mount zebrafish embryos the featured image was created from a picture of a 90% epiboly zebrafish embryo, which was stained for phospho histone h3 using a red secondary antibody and by imaging the endogenous membrane gfp in the embryo. A new zebrafish model of orointestinal pathogen colonization. Immunohistochemistry allows visualization of antigens usually proteins within an embryo. The 5th edition is available in print and within the zfin protocol wiki. Whole mount immunohistochemistry and in situ hybridization of. Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. The following whole mount tunel protocol is adapted from parsons et al. However, little staining is observable because of the relatively similar appearance between the stained somite cells and surrounding cells, due to the secondary antibody resulting in stained cells which resembled surrounding cells. Expression of maeg was analyzed by whole mount in situ hybridization and whole mount antibody staining.
The 5th edition is available in print and within the. We currently offer a continuously expanding product list for research in zebrafish. Abstract whole mount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Wrap in foil and incubate overnight at 4c on gentle rocking device. Since mouse skin is relatively thin, it is possible to image through the full thickness of flattened skin using conventional confocal microscopy. Dechorionate 26 hr embryos pharyngula stage carefully with two fine forceps. Loss of fluorescence through irreversible photobleaching processes can lead to a significant reduction in sensitivity, particularly when target molecules are of low abundance or when excitation light is of high intensity or long duration. Two zebrafish mutants that do not develop brain ventricles are snakehead and. Whole mount antibody staining of zebrafish embryos for markers of segmentation. Use of zebrafish danio rerio embryos as a model to. A certain amount of flexibility in the staining schedule is possible.
Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. Whole mount immunohistochemistry in zebrafish embryos and. Histological methods protocols zfin community wiki. Ag,ik gene expression by whole mount in situ hybridization. If the signal is strongly diminished after the methanol treatment, one can use the nonmethanol protocol, or test alternative antibodies against the target protein. To this end, an antibody based fluorescence double staining protocol for whole mount zebrafish embryos and larvae was adapted to simultaneously detect intrafollicular t3 and t4. A simple whole mount staining protocol for bone and or cartilage in adults and larvae. Visualizing multiciliated cells in the zebrafish through a combined. This video demonstrates the different steps in performing wholemount antibody staining in young chick embryos. Typically, a primary antibody binds specifically to an antigen e. Larva analyzed 3 days postinfection by wholemount immunofluorescence, using an antibody staining bacteria.
One for alla highly efficient and versatile method for. Useful tip for better zebrafish whole mount ihc staining the zebrafish danio rerio is a common vertebrate model organism in scientific research for studying vertebrate development, gene function, and regenerative abilities. Zebrafish disease models almost uniquely provide a basis for efficient whole animal. Review and cite whole mount staining protocol, troubleshooting and other methodology information contact experts in whole mount staining to get answers. Larva zstack taken with a confocal microscope and 40. Staining of 30 hpf fish or older is improved by adding triton x100 to a final concentration of 0. These can be used for imaging with a 2photon or confocal microscope. Whole mount staining of zebrafish embryos, now commonly used, requires extra steps to fix and permeabilize to. Useful tip for better zebrafish wholemount ihc staining. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Add embryos to siliconized microcentrifuge tubes no more than 100 per tube. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. Representative results of whole mount double fluorescent in situ hybridization. It involves the process of selectively identifying antigens proteins in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
This article describes methods for dissecting, fixing, labeling, and imaging mouse skin as an intact twodimensional sheet, referred to as a whole mount or flat mount preparation. In wholemount ihc, the distribution of an antigen can be mapped rapidly and reliably in the embryos. Make observations of the stained embryos in a glass petri dish do not use polystyrene dishes. Zebrafish sperm cryopreservation pdf dna injection protocol pdf bleaching zebrafish embryos. The method described herein displays a reliable and useful technique of whole mount immunostaining. If staining with more than one antibody, prepare a pool of antibodies together. One for alla highly efficient and versatile method for fluorescent. However, do not rock at high speed since the embryos are somewhat fragile. Can anyone give a procedure for whole mount sv2 staining for zebrafish larvae 120hpf can anyone recommend a protocol. Whole mount immunostaining of zebrafish embryos this protocol is effective on embryos up to 4 dpf note that deeper tissue will be much harder to stain after 32 hpf this protocol generally works for most antibodies we have tried. Transparency is a unique attribute of zebrafish that permits direct assessment of drug effects on the nervous system using whole mount antibody immunostaining and histochemistry.
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