Solovei Irina, Cremer Marion
Institute of Human Genetics, Biozentrum (LMU), University of Münich, Planegg-Martinsried, Germany.
Methods Mol Biol. 2010;659:117-26. doi: 10.1007/978-1-60761-789-1_8.
Fluorescence in situ hybridization on three-dimensionally preserved nuclei (3D-FISH), in combination with immunocytochemistry and 3D fluorescence microscopy, is a key tool to analyze the functional organization of the interphase nucleus. In the last decade, 3D-FISH on cultured cells has become a routine technique and is now widely used in nuclear biology. This method allows visualization of chromosome territories, chromosome subregions, single genes, and RNA transcripts preserving their spatial positions in the cell nucleus. In many cases, it is desirable to combine 3D-FISH and immunostaining to map DNA/RNA and protein targets in the same cells. Some steps of the FISH procedure, however, may interfere with immunostaining and special efforts have to be done to combine FISH and antibody staining successfully. The protocol suggested in this chapter describes three variants of combined 3D-FISH and immunostaining which have been successfully used in our laboratory for many years.
三维保存细胞核荧光原位杂交（3D-FISH），结合免疫细胞化学和三维荧光显微镜，是分析间期细胞核功能组织的关键工具。在过去十年中，培养细胞上的3D-FISH已成为一项常规技术，目前广泛应用于核生物学。该方法能够可视化染色体区域、染色体亚区域、单个基因和RNA转录本，同时保留它们在细胞核中的空间位置。在许多情况下，人们希望将3D-FISH与免疫染色相结合，以在同一细胞中定位DNA/RNA和蛋白质靶点。然而，FISH程序的某些步骤可能会干扰免疫染色，因此必须做出特殊努力才能成功地将FISH与抗体染色相结合。本章建议的方案描述了3D-FISH与免疫染色相结合的三种变体，这些变体在我们实验室已成功使用多年。
