Double fluorescent in situ hybridization to zebrafish embryos
Double fluorescent in situ hybridization to zebrafish embryos
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DOI：
10.1016/s0168-9525(96)90091-8
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发表时间：
1996-10-01
影响因子：
11.4
通讯作者：
Yan, YL
中科院分区：
文献类型：
--
作者：
Jowett, T;Yan, YL
描述了一种在斑马鱼组织切片或整装胚胎中使用显色底物进行双色蛋白原位杂交的方法。比较了3种碱性磷酸酶偶联抗体的荧光底物：载体红碱性磷酸酶底物(矢量实验室)、坚固红片(Boehringer Mannheim)和Sigma FastFast Red TR/NANATHOL AS-MX(Sigma)。一个探针用荧光素标记，另一个用地高辛标记，分别与碱性磷酸酶标记的荧光素和地高辛标记抗体连续孵育。斑马鱼胚胎15-16h的大脑中有hoxb2和krx20的双重荧光原位杂交图像。对提高探针杂交可视化水平的对照实验提出了建议。
A protocol for 2-colourin situhybridization using chromogenic substrates in zebrafish tissue sections or whole-mount embryos is described. Three fluorescent substrates for alkaline phosphatase-conjugated antibodies were compared: Vector Red alkaline phosphatase substrate (Vector Laboratories), Fast Red tablets (Boehringer Mannheim) and SigmaFastFast Red TR/Naphthol AS-MX (Sigma). One probe is labelled with fluorescein, and the other with digoxigenin, and probes were visualized by sequential incubation in alkaline phosphatase-conjugated antibodies to fluorescein and digoxigenin respectively. Double-fluorescentin situhybridization images are shown forhoxb2andkrx20in brains of 15-16 h zebrafish embryos. Suggestions are given for control experiments to improve visualization of probe hybridization.