Im wondering if anyone has a protocol for clickit plus tunel kit invitrogen using whole mount zebrafish 4dpf, will be looking at various brain regions. Acknowledgements 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. Nestin is essential for zebrafish brain and eye development. Severe stress conditions, such as heat shock at 39c for 1 h, ultraviolet light at 10100 mjcm 2 and ray irradiation at 520 gy induced extensive apoptosis in. Cryopreservation and in vitro fertilization ivf protocols. Whole mount staining is very similar to immunocytochemistry icc or staining of cryosections.
Tunel positive apoptotic cells were observed predominantly in the eyes, brain and spinal cord at 24 h. Stressinduced apoptosis in zebrafish embryos can be monitored by whole mount tunel staining and caspase assay. Extensive apoptosis and abnormal morphogenesis in pro. Whole mount tunel staining showed the spatial distribution of apoptotic cells in the transgenic embryos. Role of zebrafish lbx2 in embryonic lateral line development. The tunel terminal dutp nickendlabeling assay to detect cell death in zebrafish embryos was carried out as previously described, then after several washes in pbst, the embryos were transferred to dapi solution to visualize the nuclei of the pll primordium. Article analysis of apoptosis in zebrafish embryos by wholemount im. I want to detect apoptosis on zebrafish larva 35 dpf, so i conducted a tunel assay experiment following the instructions provided by the supplier online in situ cell death detection kit.
The following wholemount tunel protocol is adapted from parsons et al. Acridine orange staining and visualization in zebrafish to study. Analysis of apoptosis in zebrafish embryos by wholemount. Methodologyprincipal findings as nestin is conserved in zebrafish and its predicted. Wholemount immunofluorescence to detect activated caspase 3 casp3 assay is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. Yag lasers in the treatment of gingival melanin pigmentation. Tunel is the gold standard and still easy to do wholemount or on sections. Ad confocal 3d projections of wholeheart tunel staining of tgfli1a. Ihc staining protocol for whole mount samples abcam. 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. Protocol using clickit plus tunel assay kit in whole.
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