Kahn E, Hotmar J, Bernheim A
Institut National de la Santé et de la Recherche Médicale U.66, Institut Gustave Roussy, Villejuif, France.
Anal Quant Cytol Histol. 1996 Apr;18(2):109-14.
To determine the three-dimensional (3D) position of target sequences and chromosomal volumes in interphase human nuclei by confocal laser scanning microscopy (CLSM) by using the heterochromatin part of the long arm of human chromosome Y (HPLAHC) as a target for specific DYZ1 probes, then by D10Z1 probes specific to the centromere of chromosome 10.
Fluorescence in situ hybridization information inside chromosomal preparations was obtained with FITC-labelled probes and propidium iodide (PI) as a DNA-specific stain. To have a control in the experiment, HPLAHC Y was taken as a model of a domain and the centromere of chromosome 10 as a model of a single centromere spot. To have access to their 3D visualization, we selected FITC and PI patterns of fluorescence when optical slices were obtained and used a 3D reconstruction software.
Labelling of the target by the probes was characteristic of Y heterochromatin and chromosome 10 centromere localizations and allowed observation of their domain in the x, y and z directions.
This work was performed on two sets of 30 stained interphase nuclei. Deformations were confirmed by fluorescent spherical beads mounted in the same medium and scanned in the same conditions.
通过共聚焦激光扫描显微镜（CLSM），以人类Y染色体长臂的异染色质部分（HPLAHC）作为特异性DYZ1探针的靶标，然后以10号染色体着丝粒特异性的D10Z1探针，来确定间期人类细胞核中靶序列的三维（3D）位置和染色体体积。
使用异硫氰酸荧光素（FITC）标记的探针和碘化丙啶（PI）作为DNA特异性染色剂，获取染色体制备物中的荧光原位杂交信息。在实验中，将HPLAHC Y作为一个结构域的模型，将10号染色体的着丝粒作为单个着丝粒点的模型，以进行对照。为了实现它们的三维可视化，我们在获取光学切片时选择了FITC和PI的荧光模式，并使用了三维重建软件。
探针标记靶标呈现出Y异染色质和10号染色体着丝粒定位的特征，并能在x、y和z方向观察到它们的结构域。
这项工作是在两组30个染色的间期核上进行的。通过安装在相同介质中并在相同条件下扫描的荧光微球证实了变形情况。
