A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.
A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.
复制标题
使用多核苷酸特异性荧光染料原位可视化小鼠囊胚的内细胞团和滋养外胚层核的快速程序。
DOI：
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发表时间：
1984
期刊：
影响因子：
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通讯作者：
Susan Hunter
中科院分区：
文献类型：
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作者：
A. Handyside;Susan Hunter
用多核苷酸特异性荧光染料原位标记外滋养外胚层（TE）和内细胞团（ICM）细胞核，建立了一种快速计数小鼠囊胚外滋养外胚层和内细胞团细胞的方法。通过使用选择性抗体介导的补体裂解使细胞透化，用碘化丙啶（PI）标记TE核（Solter和Knowles，'75）。然后将胚泡固定在乙醇中，并用双苯甲亚胺标记ICM核。这两种荧光染料具有非常不同的荧光光谱。因此，通过使用荧光显微镜与适当的过滤器组合，PI标记的TE核出现粉红色或红色;双苯甲酰亚胺标记的ICM核，蓝色或未标记。通过差异标记计数的胚泡核总数和ICM核数与通过空气干燥铺展完整胚泡或免疫手术分离的ICM核后检测到的数量相似（Tarkowski '66）。TE和ICM细胞核原位差异标记有两个重要的优点-这两种细胞类型的数量可以确定单个囊胚和空间关系部分保留，以便区域相互作用可以进行研究。
A rapid procedure has been devised to count the numbers of outer trophectoderm (TE) and inner cell mass (ICM) cells of mouse blastocysts by differentially labelling their nuclei in situ with polynucleotide-specific fluorochromes. The TE nuclei were labelled with propidium iodide (PI) by permeabilising the cells using selective antibody-mediated complement lysis (Solter and Knowles, '75). The blastocysts were then fixed in ethanol and the ICM nuclei labelled with bisbenzimide. These two fluorochromes have widely different fluorescent spectra. Thus, by using fluorescence microscopy with appropriate filter combinations, the PI-labelled TE nuclei appeared pink or red; the bisbenzimide-labelled ICM nuclei, blue or unlabelled. The total numbers of blastocyst nuclei and the numbers of ICM nuclei counted by differential labelling were similar to the numbers detected after spreading the nuclei of intact blastocysts or immunosurgically isolated ICMs by air-drying (Tarkowski '66). Differential labelling of TE and ICM nuclei in situ has two important advantages--that the numbers of both these cell types can be determined for individual blastocysts and that spatial relationships are partially preserved so that regional interactions can be studied.