Stressinduced apoptosis by heat shock, uv and gammaray. Additional wholemount techniques to identify apoptotic cells in zebrafish embryos include the tunel assay8 and acridine orange ao staining9. Fertilized embryos were divided into seven groups as control, sham, and five experimental groups. Then, i apply tricaine 178 mgl for 60 seconds and view the larva under the microscope. This protocol was originally modified from klymkowski and hanken 1991 for amphibians. Cooperative interaction of angiopoietinlike proteins 1. 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. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted. Neutral red staining has been used as a visual marker of epithelial damage in zebrafish larvae mcleish et al. Environmental stressinduced apoptosis in zebrafish danio rerio embryos was characterized by assaying caspase3like activity and wholemount terminal deoxynucleotidyl nickend labeling tunel staining. Frontiers neuroprotective role of the pi3 kinaseakt. Faf1, a gene that is disrupted in cleft palate and has conserved.
Bremiller from zebrafish book 5th edition resin sections. Animals were fixed in 4% pfa in pbs for 1 hour and processed as per the protocol without proteinasek treatment. To address the effect of cc on heart apoptosis in the zebrafish we performed tunel tdtmediated nick end labeling staining on the hearts of tgfli1a. 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. E wholemount odianisidine staining of hemoglobin in control and tertknockdown embryos coinjected with either zp53mo or zp53controlmo at 48 hpf. In the present study, biological effects of elfmf on the development of zebrafish danio rerio embryos were investigated. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Despite its wide use as an npc marker, the function of nestin in embryo development is unclear. Hybridizations were performed at the indicated times using zegapspecific a or zmak3specific b digoxigenin diglabeled rna probes reaction color bluepurple. Nestin is essential for zebrafish brain and eye development. O in e3 media for 20 minutes and wash it in e3 media for 5 minutes.
Background nestin is expressed in neural progenitor cells npc of developing brain. Protocol using clickit plus tunel assay kit in whole. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. Whole mount staining is the staining of small pieces of tissue usually embryos without sectioning. This work was supported, in part, by grants from the japan society for the promotion of science, the japan science and technology corporation, and the ministry of agriculture, forestry, and fisheries of japan. For the best results, it is important to over stain heavily the wholemount preparation. Thus, zebrafish is a useful experimental model animal for investigation of apoptosis in vivo. Wholemount tunel staining of zebrafish embryos was adapted from a tissuesectioning method yabu et al.
Tunel or acridineorange staining of 1 ppb tcdd treated embryos at. Extremely lowfrequency elf magnetic field mf, as a widespread ecological factor, has an influence on all living beings. 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. Wholemount immunofluorescent analysis was performed on control ctl mo and exon 9 ex9 mo morpholinoinjected embryos at 48 hpf. This study presents data illustrating the role of pi3kakt in attainment of normal brain size during zebrafish embryogenesis. Collagen vi antibody labeling in col6a1 morphant zebrafish. Cisstilbene combretastatin a4 ca4 and a large group of its derivant compounds have been shown significant antiangiogenesis activity. Gfppositive embryos, as well as uninjected control embryos, were irradiated 15 gy at 14 hpf and analyzed by wholemount tunel staining at 20 hpf. The numbers of tunel positivecells in the retina were counted.
How to permeabilize zebrafish larva in tunel assay experiment. The following wholemount tunel protocol is adapted from parsons et al. Im wondering if anyone has a protocol for clickit plus tunel kit invitrogen using wholemount zebrafish 4dpf, will be looking at various brain regions. For whole mount in situ hybridization, embryos older than 48 hpf were incubated in egg water containing 0. Article analysis of apoptosis in zebrafish embryos by wholemount im. However the side effects even the toxicities of these. Video article analysis of apoptosis in zebrafish embryos. A chemical enterocolitis model in zebrafish larvae that is. Whole mounts were mounted in 90% glycerol pbs for micros copy and. Extensive apoptosis and abnormal morphogenesis in pro. Ctl mo embryos a demonstrated a fine, reticulated pattern of staining with their myotomes arrow. The intensity of the blood flow color over the yolk indicates the hemoglobin concentration ad. Wholemount in situ hybridization wish analysis of 2 dpf embryos with. Among these transcription factors, runx1 is a prerequisite for anteriorspecific palatal fusion.
Time course of tunel staining in wholemount and retinal sections. Anterior cleft palate due to cbfb deficiency and its. Isolated animal poles were examined in embryo rearing medium erm after incubation in erm containing ao and pi. Additional wholemount techniques to identify apoptotic cells in zebrafish embryos include the tunel assay 8 and acridine orange ao staining 9. Combretastatin a4 efficiently inhibits angiogenesis and. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Embryonic growthassociated protein is one subunit of a. Whole mount in situ hybridization and antibody staining. Faf1, a gene that is disrupted in cleft palate and has. Figure 4 identification, characterization, and effects. 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.
If not, the tongue prevents the palate from closing, resulting in. This study showed transient high levels of apoptosis in various cells and tissues during specific phases of development 16, 18. Embryos reaching the desired developmental stages were fixed in 4% pfapbs. We currently offer a continuously expanding product list for research in zebrafish. Our assays dispute earlier observations that immersion in tnbs does not cause a chemical burn on surface epithelial layers when used at a. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. At 24hpf, we detected apoptotic cells in different regions of the developing zebrafish embryos, such as in the region of the eye, the otic vesicle and the developing brain. Wholemount terminal deoxynucleotidyl transferasemediated dutp nickend labelling tunel staining and immunostaining. Cbfb is a cofactor of the runx family of transcription factors.
Would someone have a detailed edu labelling protocol for. Consistent with this, markedly elevated tunel staining in the midbrain and hindbrain. A simple wholemount staining protocol for bone and or. Zebrafish models of collagen virelated myopathies human. Visualization of both tunel staining and antiactive caspase3 staining section 2. Zebrafish whole mount immunohistochemistry youtube.
Mechanisms of fasdassociated defects are not well understood. Tunel staining showed more apoptosis in the neural plate and tube and in. Embryos were fixed overnight at 4c in a 4% solution of formaldehyde in phosphatebuffered saline pbs. Tunel staining in whole mount hydractinia echinata using in situ brdured dna fragmentation tunel assay kit ab66110. A representative region of tunel positive cells is indicated by the brackets c, d. The wholemount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning andor. Make observations of the stained embryos in a glass petri dish do not use polystyrene dishes. Genetex is pleased to introduce our new line of zebrafish antibodies with true validation.
Citeseerx document details isaac councill, lee giles, pradeep teregowda. Zebrafish wholemount in situ hybridization and alcian blue. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. If an antibody has been used successfully on cryosections this does not include paraffinembedded sections, then the antibody should work for. The patterns of tunel staining and cleaved caspase3. Positive tunel signals were detected with an abc kit elite, vector. Although you can stain both cartilage and bone in the same specimen, you need to be aware that bone staining can mask cartilage staining, for instance when perichondral ossification takes place around a cartilage template. Atg, tunel staining showed more apoptosis in the neural plate and tube and in the. Stressinduced apoptosis in zebrafish embryos can be monitored by whole mount tunel staining and caspase assay. We present a method to visualize apoptotic cells in living zebrafish by a genetically encoded marker. In place of proteinasek animals were permeabilised in 3% triton in pbs for 15 minutes. Zebrafish wholemount in situ hybridization and alcian blue staining. Louis, mo, usa from 12 hpf onwards to prevent pigmentation. Zebrafish embryos were stained with alcian blue according to the method of kelly and bryden.
Using tunel staining in wholemount embryos, no patterns of localized cell death were apparent from 17 to 24 hpf fig. Neuronal survival and growth in the embryo is controlled partly by trophic factors. Analysis of apoptosis in zebrafish embryos by wholemount. Immerse slides briefly 30 to 90 sec in the staining solution, wash in dh2o, and dehydrate as above. This study used the zebrafish danio rerio as a vertebrate. The cells labeled using seca5yfp show several characteristics of apoptotic cells. The occurrence of apoptotic cells during the first 4 d of zebrafish embryonic development has been studied and quantified previously by using in situ tunel 17. 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. Add 1 g powder to 66 ml to glycerol and heat at 60 0 c for one hour. 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. Wholemount in situ hybridization showing spatial and temporal expression patterns of zegap and zmak3 mrnas in the developing zebrafish. Suppression of apoptosis by bcl2 overexpression in. Retinal neuronal cell is a toxicological target of.
Despite detailed in vivo knowledge of glycolytic enolases and many bacterial nonenolase members of the superfamily, little is known about the in vivo function of vertebrate nonenolase enolase superfamily members enosf1s. Sections of wholemount in situ hybridization preparations. Ihc staining protocol for whole mount samples abcam. Fetal alcohol syndrome fasd describes a range of birth defects. We have used all three methods to identify apoptotic cells in zebrafish embryos 4,6,10. Results of previous studies suggest involvement of the. Role of zebrafish lbx2 in embryonic lateral line development. Neurotoxicity assessment using zebrafish by chuenlei. Egfpbcl2expressing embryos had fewer apoptotic cells than gfpexpressing or uninjected control fish n 12 per treatment group following irradiation treatment p.
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