Extensive apoptosis and abnormal morphogenesis in procaspase3. Caspases and other apoptosisregulating genes have been cloned from zebrafish danio rerio and other fish species. 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. Apoptosis in zebrafish whole mounts was detected by tunel using in situ. Article analysis of apoptosis in zebrafish embryos by wholemount im.
Ihc staining protocol for whole mount samples abcam. Embryos were fixed overnight at 4c in a 4% solution of formaldehyde in phosphatebuffered saline pbs. No staining was present in embryos hybridized with sense probe 256 cell and. Df basolateral nakatpase is present in the 74 hpf anterior intestinal epithelium, but there is minimal staining in the posterior intestine. Stressinduced apoptosis by heat shock, uv and gray. A guide for the laboratory use of zebrafish danio rerio. A wholemount, interior view of the adult zebrafish anterior. End labeling tunel staining in embryonic and adult brains of biluo mutants. The zebrafish book a guide for the laboratory use of zebrafish danio brachydanio rerio by monte westerfield, institute of neuroscience, university of oregon. C and d, g and h tunel staining showing apoptotic cells in the hindbrain of a representative. Environmental stressinduced apoptosis in zebrafish danio rerio embryos was. The zebrafish homologue of nedd1 znedd1 was cloned and found to have a similar.
Environmental stressinduced apoptosis in zebrafish danio rerio embryos was char acterized by. For the best results, it is important to over stain heavily the wholemount preparation. These features include condensed chromatin, tunel staining, blebbing, and fragmentation of the cells. Developmental apoptosis mediates entry and positioning of. Bremiller from zebrafish book 5th edition resin sections. Compared to acridine orange staining, which can identify apoptotic cells in live. This antibody was not suitable for wholemount staining, and hence the. Wholemount tunel staining of zebrafish embryos was adapted from a tissue sectioning method yabu et al. A guide for the laboratory use of zebrafish danio brachydanio rerio. Dysregulation of microglial function contributes to. Published procedures for the wholemount tunel staining of embryos were. Extensive apoptosis and abnormal morphogenesis in pro.
An essential function for the centrosomal protein nedd1 in. 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. Activation of apoptosis and caspasea3 in zebrafish early gastrulae. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The cells labeled using seca5yfp show several characteristics of apoptotic cells. Use embryos in which alkaline phosphatase has been employed as a detection enzyme. Sections of wholemount in situ hybridization preparations. How to permeabilize zebrafish larva in tunel assay experiment.
Wholemount tunel staining of zebrafish em bryos was. We present a method to visualize apoptotic cells in living zebrafish by a genetically encoded marker. Analysis of apoptosis in zebrafish embryos by wholemount immunofluorescence to. Analysis of apoptosis in zebrafish embryos by wholemount. T tunel assay identifies apoptotic cells at the tips of intestinal folds. Intestinal growth and differentiation in zebrafish sciencedirect. Zebrafish noxa promotes mitosis in early embryonic development. In this study, we have studied the role of zebrafish noxa znoxa by using. Acridine orange staining is nice for a quick and dirty test of cell death, but tunel is the gold standard and still easy to do wholemount or on sections. Wholemount tunel staining of zebrafish embryos was adapted from a tissuesectioning method yabu et al. Apoptosis programmed cell death is important in normal biological processes and in pathogenesis in vertebrates. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections. Whole mount staining of biotindextran injected zebrafish embryos photoconversion of.
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