Institut Curie, CNRS UMR3215/ INSERM U934, Paris Sciences & Lettres Research University (PSL), Paris, France.
Institut de Génétique Moléculaire de Montpellier, Univ Montpellier, CNRS, Montpellier, France.
Methods Mol Biol. 2021;2214:157-173. doi: 10.1007/978-1-0716-0958-3_11.
Immunofluorescence and RNA fluorescence in situ hybridization (FISH) methods enable the detection of, respectively, proteins and RNA molecules in single cells. Adapted to preimplantation mouse embryos, these techniques allow the investigation of transcriptional dynamics in the first embryonic and extraembryonic lineages and can circumvent the limited amount of starting material. This can as well be coupled to examination of chromatin modification, i.e., histone marks, by immunofluorescence. Here is outlined an immunofluorescence protocol combined to nascent RNA-FISH after immunosurgery of the mouse inner cell mass of the blastocyst to study early changes in transcription and/or histone marks of both primitive endoderm and epiblast cells. The method describes the different steps from coverslips and FISH probe preparation to inner cell mass isolation and immunofluorescence followed by RNA-FISH. Furthermore, this is applicable to earlier developmental stages and other mammalian species provided little technical adjustments.
免疫荧光和 RNA 荧光原位杂交（FISH）方法分别能够检测单个细胞中的蛋白质和 RNA 分子。将这些技术应用于着床前的小鼠胚胎，可以研究第一胚胎和胚胎外谱系中的转录动态，并且可以避免起始材料的数量有限。这也可以与免疫荧光检查染色质修饰（即组蛋白标记）相结合。这里概述了一种免疫荧光方案，该方案与免疫手术结合使用，对囊胚的内细胞团进行免疫手术，以研究原始内胚层和上胚层细胞中转录和/或组蛋白标记的早期变化。该方法描述了从盖玻片和 FISH 探针制备到内细胞团分离以及随后进行免疫荧光和 RNA-FISH 的不同步骤。此外，该方法适用于更早的发育阶段和其他哺乳动物物种，只需进行一些技术调整。
