Handyside A H, Hunter S
J Exp Zool. 1984 Sep;231(3):429-34. doi: 10.1002/jez.1402310317.
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.
已设计出一种快速程序，通过用多核苷酸特异性荧光染料原位差异标记小鼠囊胚的细胞核来计数外滋养层（TE）和内细胞团（ICM）细胞的数量。使用选择性抗体介导的补体裂解使细胞通透，从而用碘化丙啶（PI）标记TE细胞核（索尔特和诺尔斯，1975年）。然后将囊胚固定在乙醇中，并用双苯甲酰胺标记ICM细胞核。这两种荧光染料具有广泛不同的荧光光谱。因此，通过使用配备适当滤光片组合的荧光显微镜，PI标记的TE细胞核呈现粉红色或红色；双苯甲酰胺标记的ICM细胞核呈现蓝色或未标记。通过差异标记计数的囊胚细胞核总数和ICM细胞核数与通过空气干燥完整囊胚或免疫手术分离的ICM细胞核后检测到的数量相似（塔尔科夫斯基，1966年）。原位差异标记TE和ICM细胞核有两个重要优点——可以确定单个囊胚中这两种细胞类型的数量，并且部分保留了空间关系，以便研究区域相互作用。
