Reguzzoni M, Protasoni M, Maserati M, Pressato B, Manelli A, Raspanti M
Department of Human Morphology L. Cattaneo, University of Insubria, Varese, Italy.
Arch Histol Cytol. 2005 Jun;68(2):115-20. doi: 10.1679/aohc.68.115.
The hybridization site of a DNA probe was detected using a scanning electron microscope (SEM), modifying the standard in situ hybridization (ISH) method. The experiments were performed on human metaphases obtained from lymphocyte cultures of human peripheral blood. The libraries and probes used were: 1-chromosome library for the painting of chromosome 1 (wcp 1), an alphoid centromere-specific probe of chromosome 8 (pZ8.4), and the yeast artificial chromosome (YAC) 964-C10 mapped at band p13 on chromosome 12. These probes were labeled by nick translation with biotin and displayed with a gold-conjugated anti biotin goat antibody. The gold signal was amplified by silver enhancement. The chromatides appeared as packages of thin filaments 120 nm high; some of them collapsed, probably due to ISH procedures. All the probes were clearly detected as small gold particles grouped on the surface of the target chromosomes and chromosome sites. Thus, this procedure is useful to clarify the positional relationship between the chromatin filaments and the probe.
采用扫描电子显微镜（SEM）检测DNA探针的杂交位点，对标准原位杂交（ISH）方法进行了改进。实验在取自人外周血淋巴细胞培养物的人类中期染色体上进行。使用的文库和探针有：用于染色体1描绘的1号染色体文库（wcp 1）、染色体8的α卫星着丝粒特异性探针（pZ8.4）以及定位在染色体12 p13带的酵母人工染色体（YAC）964 - C10。这些探针通过缺口平移法用生物素标记，并用金偶联的抗生物素山羊抗体显示。金信号通过银增强进行放大。染色单体呈现为120纳米高的细丝束；其中一些可能由于ISH程序而塌陷。所有探针均清晰地检测为聚集在目标染色体和染色体位点表面的小金颗粒。因此，该程序有助于阐明染色质细丝与探针之间的位置关系。
