A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.
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.
D000818:Animals; D006690:Bisbenzimidazole; D001755:Blastocyst / Q000166:cytology*; D002467:Cell Nucleus / Q000648:ultrastructure; D005260:Female; D005456:Fluorescent Dyes; D051379:Mice; D008856:Microscopy, Fluorescence; D011119:Polynucleotides; D011247:Pregnancy; D011419:Propidium; D014327:Trophoblasts / Q000648:ultrastructure*
D005456:Fluorescent Dyes; D011119:Polynucleotides; D011419:Propidium; D006690:Bisbenzimidazole