早期鸡胚的双重整体原位杂交
Double whole mount in situ hybridization of early chick embryos.
作者信息
Psychoyos Delphine, Finnell Richard
机构信息
Center for Environmental and Genetic Medicine, Institute of Biosciences and Technology, Texas A&M Health Science Center, TX, USA.
出版信息
J Vis Exp. 2008 Oct 27(20):904. doi: 10.3791/904.
The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC. Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for photograph and sectioning. A troubleshooting guide is also presented.
鸡胚是研究早期胚胎发育的宝贵工具。它的透明性、易获取性和易于操作的特点，使其成为研究大脑、神经管、体节和心脏原基形成过程中基因表达的理想工具。本视频展示了双色全胚胎原位杂交的不同步骤；首先，将胚胎从卵中取出并固定在多聚甲醛中。其次，对胚胎进行预杂交处理。然后将胚胎与两种不同的探针杂交，一种与地高辛（DIG）偶联，另一种与异硫氰酸荧光素（FITC）偶联。过夜杂交后，将胚胎与地高辛偶联抗体孵育。进行地高辛底物的显色反应，感兴趣的区域呈现蓝色。然后将胚胎与异硫氰酸荧光素偶联抗体孵育。对胚胎进行异硫氰酸荧光素的显色反应，感兴趣的区域呈现红色。最后，将胚胎固定并进行拍照和切片处理。还提供了一份故障排除指南。
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