The patterns of tunel staining and cleaved caspase3. Time course of tunel staining in wholemount and retinal sections. Neuronal survival and growth in the embryo is controlled partly by trophic factors. 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 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. Sections of wholemount in situ hybridization preparations.
Procaspase3 transgenic zebrafish exhibited abnormal morphogenesis in the eyes. Function and regulation of auts2, a gene implicated in. To detect apoptotic cells induced by copper we incubated 72 hpf larvae with 300 m copper for 2 h. A for the detection of primitive hematopoietic markers, 20somite stage zebrafish embryos 19 hpf are shown and are oriented. Bremiller from zebrafish book 5th edition resin sections. Rip3 knockdown rescues photoreceptor cell death in blind. Angiopoietinlike protein angptl 1 and angptl2, which are considered orphan ligands, are highly homologous, particularly in the fibrinogenlike domain containing the putative receptor binding site. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections.
Here we describe a technique that employs in vitro synthesized rna tagged with digoxigenin uridine5. Whole mount immunohistochemistry is a valuable tool for the study of spatial and temporal protein expression during zebrafish development. Research paper yesassociated protein yap is required. Extensive apoptosis and abnormal morphogenesis in procaspase3. Environmental stressinduced apoptosis in zebrafish danio rerio embryos was characterized by assaying caspase3like activity and wholemount terminal deoxynucleotidyl nickend labeling tunel staining. After 60 minutes of staining embryos were washed three times in e3 media.
However, phenotypic analysis often does not go beyond establishing overall distribution. Stressinduced apoptosis in zebrafish embryos can be monitored by whole mount tunel staining and caspase assay. In place of proteinasek animals were permeabilised in 3% triton in. Extensive apoptosis and abnormal morphogenesis in pro. Positive tunel signals were detected with an abc kit elite, vector. Fix the fish in 10% neutral buffered formalin for a few hours to one day depending on the size of the specimen. Hematopoiesis is an orderly but complex dynamic process that includes differentiation, development, and maturation of hematopoietic stem and progenitor cells hspcs controlled by many factors. Collagen vi antibody labeling in col6a1 morphant zebrafish. Histological analyses of more than 90 zebrafish of each genotype derived from 17 different clutches confirmed that the testicular phenotype described herein was correlated with homozygous brca2 q658x mutation in all cases, and was maintained after three outcrosses to wt zebrafish. Uv radiation and zebrafish biology bibliographies cite.
This similarity suggests potentially cooperative functions between the two proteins. C and d, g and h tunel staining showing apoptotic cells in the hindbrain of a representative. Wholemount in situ hybridization of control and tert deficient zebrafish embryos at the 20somite stage 19 hpf a, and at 2832 hpf b. Whole mount immunohistochemistry in zebrafish embryos and. This was performed almost exactly as described in at a the zebrafish book dilution of 1. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Wholemount antibody labeling of 48 hpf control embryos with an antibody to collagen vi revealed a fine reticulated pattern in the somatic compartment arrow, fig. O in e3 media for 20 minutes and wash it in e3 media.
Sublethal concentrations of waterborne copper induce. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor. For most trophic factors such as insulinlike growth factor1, the ability to regulate cell survival has been attributed to the phosphoinositide 3kinase pi3kakt kinase cascade. Cooperative interaction of angiopoietinlike proteins 1. Cadherin4 plays a role in the development of zebrafish. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. Citeseerx document details isaac councill, lee giles, pradeep teregowda. Im wondering if anyone has a protocol for clickit plus tunel kit invitrogen using wholemount zebrafish 4dpf, will be looking at various brain regions. Stressinduced apoptosis by heat shock, uv and gammaray irradiation in zebrafish embryos detected by increased caspase activity and wholemount tunel staining. Zebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Make observations of the stained embryos in a glass petri dish do not use polystyrene dishes. We have used all three methods to identify apoptotic cells in zebrafish embryos 4,6,10.
Whole mounts were mounted in 90% glycerol pbs for micros copy and. Compared to acridine orange staining, which can identify apoptotic. Thus, zebrafish is a useful experimental model animal for. Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. Quantification of cell death demonstrated that, while more death occurred at 17 hpf than at 21 hpf, no brain region examined displayed a significant difference in amount of cell death relative to other. Article analysis of apoptosis in zebrafish embryos by wholemount im. This gives a clearer idea of where the target protein of interest is expressed within the. Using tunel staining in wholemount embryos, no patterns of localized cell death were apparent from 17 to 24 hpf fig. Zebrafish models of collagen virelated myopathies human. In situ hybridization on wholemount zebrafish embryos and. Live zebrafish embryos were stained with the vital dye acridine orange ao in order to determine the amount of apoptotic cells per embryo. Ihc staining protocol for whole mount samples abcam. In this report, the function of angptl1 and angptl2 is analyzed by using morpholino antisense technology in. Stress response and apoptosis in zebrafish embryos springerlink.
Cpp32, a novel human apoptotic protein with homology to caenorhabditis. This study presents data illustrating the role of pi3kakt in attainment of normal brain size during zebrafish embryogenesis. Wholemount tunel staining of zebrafish embryos was adapted from a tissuesectioning method yabu et al. A novel highthroughput method to detect anxietyrelated behaviors in. Since the introduction of terminal deoxynucleotidyl transferasedutp nick end labeling tunel assay in 1992, 1 the tunel assay is the most widely used in situ test for apoptosis study. Bcas2 is essential for hematopoietic stem and progenitor. The following wholemount tunel protocol is adapted from parsons et al. Identification, characterization, and effects of xenopus. 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. Wholemount tunel staining of zebrafish embryos was adapted from a. The larvae were assayed for wholemount terminal deoxynucleotidyl transferasemediated dutp nickend labeling tunel. We seek manuscripts presenting work done at all levels of biological. Retinal neuronal cell is a toxicological target of.
The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. Embryos were fixed overnight at 4c in a 4% solution of formaldehyde in phosphatebuffered saline pbs. Highresolution in situ hybridization to wholemount. The journal is an international forum for the exchange of novel and substantive information on mechanisms that control development. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. Eviscerate the specimen removing internal organs being careful not to damage the ribs. Fetal alcohol syndrome fasd describes a range of birth defects. Zebrafish exhibit spontaneous contractions mediated by gap junctions beginning at before 19 hpf before motor neuron contact 19. Isolated animal poles were examined in embryo rearing medium erm after incubation in erm containing ao and pi. Zebrafish embryos were maintained as described in the zebrafish book westerfield, 2000 in accordance with university of akron, indiana university and university of michigan policies on animal care and use. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo.
The zebrafish neuromuscular junction, like other vertebrates, is mediated by acetylcholine acting at nicotinic acetylcholine receptors. For the best results, it is important to over stain heavily the wholemount preparation. Wholemount immunofluorescent analysis was performed on control ctl mo and exon 9 ex9 mo morpholinoinjected embryos at 48 hpf. A simple wholemount staining protocol for bone and or.
This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. The embryos were then rinsed with e3 copperfree medium and maintained for 24 or 72 h and fixed. For tunel staining, embryos were transferred to methanol for 30 minutes followed by rehydration in methanolpbst pbs with 0. Background the zebrafish ladybird homeobox homologous gene 2 lbx2 has been suggested to play a key role in the regulation of hypaxial myogenic precursor cell migration. Mechanisms of fasdassociated defects are not well understood. How to permeabilize zebrafish larva in tunel assay experiment.
The numbers of tunel positivecells in the retina were counted. Tunel staining for neuronal apoptosis in only 1268 fn10a mutants was. Zebrafish embryos were maintained as described in the zebrafish book westerfield, 2000 in accordance with university of akron, indiana university, and university of michigan policies on animal care and use. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. Role of zebrafish lbx2 in embryonic lateral line development. Unlike their lbx counterparts in mammals, the function of teleost lbx genes beyond myogenesis during embryonic development remains unexplored. Representative images of wholemount eyes and corresponding sections at the. Antibody labeling of exon 9 morphant embryos, on the other hand, gave an abnormal pattern of staining. Embryos for wholemount immunocytochemistry or in situ hybridization were raised in ptu 1phenyl2 thiourea, 0. Analysis of apoptosis in zebrafish embryos by wholemount. Video article analysis of apoptosis in zebrafish embryos. A guide for the laboratory use of zebrafish danio rerio 4th ed ed.
Stress response and apoptosis in zebrafish embryos. Using acridine orange to measure cell death in ethanol. Wholemount tunel staining of zebrafish em bryos was. Whole mount immunohistochemistry and in situ hybridization. Frontiers neuroprotective role of the pi3 kinaseakt.
Principal findings abrogation of lbx2 function using a specific independent. Developmental apoptosis mediates entry and positioning of. Animals were fixed in 4% pfa in pbs for 1 hour and processed as per the protocol without proteinasek treatment. Protocol using clickit plus tunel assay kit in whole. Ctl mo embryos a demonstrated a fine, reticulated pattern of staining with their myotomes arrow. F wholemount in situ hybridization revealed that eftud2 was broadly. Activation of apoptosis and caspasea3 in zebrafish early gastrulae. Gfp transgenic zebrafish, in which the promoter of the endothelial marker fli1a drives the expression of gfp in the coronary vessels and endocardium lawson and weinstein, 2002. 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. Developmental dynamics provides a focus for communication among developmental biologists who study the progressive and dynamic emergence of form and function during embryonic development.
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