Briefly, fixed samples were incubated with blocking solution 1% bsa, 5% lamb serum, pbst, 0. Staining wholemount embryos with pap solutions for antibody staining protocols wholemount staining of biotindextran injected zebrafish embryos. Actin dynamics 1 adherens junction dynamics 16 alzheimers disease 12 ampk signaling 10 angiogenesis 44 apoptosis regulation 44 b cell receptor signaling 47 death receptor signaling 5 erbbher signaling 51 esc pluripotency and differentiation 47 g1s checkpoint 11 gpcr signaling to mapks 34 growth and differentiation control by mapks 55 hedgehog signaling 1 histone. Useful tip for better zebrafish wholemount ihc staining. Dechorionate 26 hr embryos pharyngula stage carefully with two fine forceps. The hybridomas are stored at the monoclonal antibody facility of the uo under the supervision of dr. Antibody labeling protocols approved iacuc animal use protocols cryopreservation and in vitro fertilization ivf protocols. Browse our extensive catalog for polyclonal, monoclonal, and conjugated antigfp antibodies. The monoclonal antibodies were generated with balbc mice against adult zebrafish protein, and clones were selected based on the specific staining patterns obtained with particular clones.
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. Due to their striking genomic similarity to humans, regenerative abilities, and shortened life cycle, zebrafish have become an ideal tool for studying developmental processes, gene expression, and underlying mechanisms of cancer and other diseases. 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. Whole mount staining of biotindextran injected zebrafish.
Zirc protocols zirc public wiki zebrafish international resource. This collection of sections through zebrafish embryos at four different stages of development is thought to provide some help to understand how the zebrafish embryo looks inside. The zebrafish danio rerio has emerged as a powerful model for the study of. This protocol is essentially used for the spatial and temporal characterization of novel. Eviscerate the specimen removing internal organs being careful not to damage the ribs. D oblique section through the surface of the optic nerve of an adult. Validated research applications for gfp antibodies include elisa, electron microscopy, flow cytometry, cytof. Zebrafish are increasingly used as a model system in basic, biomedical, and.
It was developed by biomedical researchers in the early 1980s to detect and localize the presence or absence of specific dna. Whole mount antibody staining was performed as described previously. Zebrafish vasa rna but not its protein is a component of the. Wholemount in situ hybridization wish is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. After washing with pbs at 4c for days, the fish were either directly subjected to wholemount antibody staining 4 dpf and 15 dpf fish or dissection 1. Myelin basic protein mbp is a major constituent of the myelin sheath of oligodendrocytes and schwann cells in the central nervous. Fix for 1 hour, wash in pbs 1% triton then permeabilize the egg membrane in in ice cold acetone pbs for 8 minutes only. Due to their striking genomic similarity to humans, regenerative abilities.
Method for whole mount antibody staining in chick protocol. Immunostaining of dissected zebrafish embryonic heart. Whole mount staining of zebrafish embryos requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilised. As a result, lateral or dorsal views of whole mount stained embryo specimens can only. Immunohistochemistry of wholemount and sectioned zebrafish tissue. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Whole mount immunohistochemistry in zebrafish embryos and. If the signal is strongly diminished after the methanol. Antifish antibody products listed below are antifish antibodies from multiple suppliers. 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. Fish is a reported alias name for the human gene sh3pxd2a, or sh3 and px domains 2a.
However, do not rock at high speed since the embryos are somewhat fragile. 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. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. Zebrafish danio rerio, a minnowlike freshwater fish, has emerged as a popular animal model for research and development. 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. Zebrafish whole mount immunohistochemistry youtube. Embryos were rehydrated, permeabilized with proteinase k, and fixed again with 4% pfapbs. Temporalspatial resolution fate mapping reveals distinct. Whole mount staining of biotindextran injected embryos. Wholemount immunofluorescence university of louisville. The increase in zebrafish genomic resources together with more. However, to further sharpen the signaltonoise ratio for each antibody requires tedious protocol optimization. Treat with secondary antibody diluted with pbsbsadmso overnight at 4c or. Clearing and staining for larval fish cartilage and bone.
Analysis of protein expression and localization by zebrafish immunostaining ihc and if, in sections or wholemount preparations ihcwm, is easy to perform with zebrafish research primary antibodies. Antivasa antibodydepleted serum fails to detect both the 80kd western band and primordial germ cells in. All the samples were directly fixed in 4% pfa at 4c for 12 days. It is recommended that as incubations can be very long in whole.
The preimmune rabbit serum does not crossreact with fish proteins of similar size. We also present a simple enzymatic method to isolate whole brains from fixed zebrafish for immunocytochemistry. Nov 29, 2012 genetex is pleased to introduce our new line of zebrafish antibodies with true validation. General wholemount immunohistochemistry of zebrafish. 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. Samples were incubated with primary antibody in blocking solution overnight at 4 c. This material is from the 4th edition of the zebrafish book. This collection of sections through zebrafish embryos at four different stages of development is thought to provide some help to. Pdf method for whole mount antibody staining in chick. Fixatives used in localizing protein epitopes in ciliated cells of zebrafish embryos have included 4% pfapbs and dents fixative, which is a mixture of methanol and dmso. 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. General wholemount immunohistochemistry of zebrafish danio.
Immunofluorescent analysis of caveolin 3 in c2c11 cells. Cells were probed without control or with a caveolin 3 polyclonal. Whole mount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. We analyzed the whole mount bone staining with alizarin red s sakatahaga et al.
Whole mount antibody labeling was performed using standard methods westerfield, 2000. We observed normal bone morphology at 0 ppm cd in 3monthold and 12monthold female and male medaka fig. 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. Dec 17, 2012 lateral views ad of meoh control a and c and carbaryltreated embryos b and d wholemount antibodystained for the znp1 antibody showing motor axons at 26 hpf a and b and 32 hpf c and d. Representative results of whole mount double fluorescent in situ hybridization. Whole mount immunohistochemistry in zebrafish embryos. C crosssection through a 3d larvae eye labeled with antipax6 antibody brown and a conespecific antibody, fret43 black. Mar 25, 2009 representative results of whole mount double fluorescent in situ hybridization. Neurotoxicity assessment using zebrafish sciencedirect. We analyzed the wholemount bone staining with alizarin red s sakatahaga et al. The staining obtained using the rnascope method appeared to be more distinct and to reflect more accurately the subcellular localization of the transcripts figure 2h, compared to the. Representative imaging of wholemount immunohistochemistry of zebrafish danio rerio. Immunostaining phosphoepitopes in ciliated organs of whole.
Whole mount antibody staining of zebrafish embryos for markers of segmentation. Whole mount immunohistochemistry in zebrafish embryos and larvae. I already used ha antibody from roche, 3f10 anti rat monoclone. Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. Add primary antibody at the required dilution concentration.
Zebrafish whole mount highresolution double fluorescent in. Show only zirc antibodies show all results per page the zebrafish information network. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. The zebrafish neuromuscular junction, like other vertebrates, is mediated by acetylcholine acting at nicotinic acetylcholine receptors. Histology protocols protocolonline histology protocols biovisa betagal staining whole mount embryos karl j clark detection of. Galactosidase and alkaline phosphatase activities in tissue harvard medical embedding and sectioning of embryos. Twocolor fluorescent in situ hybridization in the embryonic.
A simple wholemount staining protocol for bone and or. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Wholemount staining of biotindextran injected embryos. Zebrafish embryos were fixed two hours in 4% paraformaldehyde pfaphosphatebuffered saline pbs, washed, dehydrated in methanol, and stored at 20degreesc. Flat mount preparation for observation and analysis of zebrafish. Immunostaining of dissected zebrafish embryonic heart ncbi nih. We currently offer a continuously expanding product list for research in zebrafish.
For neuronal staining i use tuj1 from covance mouse anti biii tubulin 1. Wholemount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. Whole mount immunohistochemistry and in situ hybridization of. Zebrafish antibodies primary antibodies sigmaaldrich. Analysis of apoptosis in zebrafish embryos by wholemount. 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. Images show a single confocal section through the posterior region of a zebrafish embryo at the tensomite stage. If the signal is strongly diminished after the methanol treatment, one can use the nonmethanol protocol, or test alternative antibodies against the target protein. For comparison to the novel electroimmunostaining, we performed whole mount staining with protocols described by grzanna et al. 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. Ish and wholemount immunohistochemistry sullivanbrown et al. Transfer embryos using pasteur pipette with the end cut off to a 2 ml tube.
The zebrafish book, a guide for the laboratory use of zebrafish danio rerio. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a. Zebrafish embryos were fixed two hours in 4% paraformaldehyde pfaphosphatebuffered saline pbs, washed, dehydrated in methanol, and. A simple whole mount staining protocol for bone and or cartilage in adults and larvae. Zebrafish whole mount immunohistochemistry reagents. General wholemount immunohistochemistry of zebrafish danio rerio embryos and larvae protocol chapter in methods in molecular biology clifton, n.
Antivasa antibodydepleted serum fails to detect both the 80kd western band and primordial germ cells in wholemount antibody staining data not shown. Actin dynamics 1 adherens junction dynamics 16 alzheimers disease 12 ampk signaling 10 angiogenesis 44 apoptosis regulation 44 b cell receptor signaling 47 death receptor signaling. Ihc staining protocol for whole mount samples abcam. Anatomy of the 24, 48, 72 and 120 hours zebrafish danio rerio embryo. Transfected cells in the zebrafish brain are amenable to in vivo time lapse imaging. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. Whole mount immunohistochemistry nonfluorescent every incubation and wash step should be done on a rocking platform. D oblique section through the surface of the optic nerve of an adult zebrafish labeled with anti pax2 antibody in yellow and anticytokeratin antibody in green and visualized using confocal microscopy.
Explants containing transfected neurons can be cultured for in vitro analysis. Histology and immunohistochemistry online protocols. If an antibody has been used successfully on cryosections this does not include paraffinembedded sections, then the antibody should work for. Incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development will need to be much longer than normal immunocytochemistry. Betagal staining whole mount karl clark betagal staining allows identification of embryonic tissuescells expressing lacz marker protein by development of pigmented. Ijms free fulltext nitric oxide reverses the position of. The effects of carbaryl on the development of zebrafish. Anti fish antibody products listed below are anti fish antibodies from multiple suppliers.
The description of these protocols is followed by the outline of a quick decalcification method and the procedure for embedding in epoxy resin to obtain serial sections. Zebrafish immunohistochemistry springer nature experiments. Histological methods protocols zfin community wiki. Caveolae marker antibodies thermo fisher scientific us. Aug 15, 2014 the staining obtained using the rnascope method appeared to be more distinct and to reflect more accurately the subcellular localization of the transcripts figure 2h, compared to the staining in the standard and the optimized whole mount fish, which appears more aggregated figure 2d and more diffuse figure 2f. Pdf a rapid and nondestructive protocol for wholemount. A guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. This video demonstrates the different steps in performing wholemount antibody staining in young chick embryos. Fix embryos in 4% paraformaldehydepbs at 4oc overnight. The arrow indicates points of truncation and the arrowhead indicates excessive branching.
It is recommended that as incubations can be very long in whole mount staining, the antibody should be diluted in blocking buffer containing 0. A novel protocol of whole mount electroimmunofluorescence. Mbp antibody staining protocol for immunohistochemistry. Wash embryo with pbs twice for 1020 minutes each at rt. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. We conclude that the antivasa antibody is specific for vasa protein.
The transcription factor bach1 suppresses the developmental. Whole mount in situ hybridization wish is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. In performing flat mounts on wish zebrafish embryos with the protocol described in parts 15. The whole mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to. Photoconversion of fluorescently labeled profiles for em. Images show a single confocal section through the posterior region of a zebrafish embryo at the ten. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation. Electroporationbased methods for in vivo, whole mount 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.
Thin section in araldite were stained with methylene blue. Lateral views ad of meoh control a and c and carbaryltreated embryos b and d wholemount antibodystained for the znp1 antibody showing motor axons at 26 hpf a and b and 32. If the antibody yields a good signal to noise ratio, the antibody is then compatible with the methanol treatment and should work well in wholemount. Whole mount antibody staining of zebrafish embryos for. Whole mount staining of zebrafish embryos, now commonly used, requires extra steps to fix and permeabilize to ensure the egg membrane is permeabilized. Dilution varies per antibody, determine empirically for each antibody. Fix the fish in 10% neutral buffered formalin for a few hours to one day depending on the size of the specimen. This video demonstrates the different steps in performing whole mount antibody staining in young chick embryos.