一种高效的人精子核原位杂交前处理技术

目的建立高效的荧光原位杂交（fluorescence in situ hybridization,FISH）前人精子核去凝集方法和监测手段。方法精子标本经10mmol/L二硫代苏糖醇（dithiothreitol,DTT）处理,使人精子核去凝集,倒置相差显微镜监测精子核膨胀度。标本固定后制片,变性后行FISH分析。结果倒置相差显微镜下,人精子核膨大程度清晰可见,膨大精子细胞边缘清楚并完整。荧光显微镜下,可见精子头部染成蓝色,边缘清楚,背景清晰,精子杂交信号清晰,非特异性杂交少。结论本方法具有简便、高效的特点,精子核解聚程度可控性和可见性强,杂交后荧光信号强、易分辨和非特异性性杂交率低,可用于人精子核FISH前处理。

Establishment of an effective approach to the treatment of human sperm nucleus before fluorescence in situ hybrization

Objective： To develop an effective treatment method of human sperm nucleus before fluorescence in situ hybrization （FISH）. Methods： The human sperm nucleus were treated with 10mmol/L dithiothreitol （DTT） to render them accessible to the DNA probes. The inverted microscope was used to control the expansion of the human sperm nucleus. FISH was performed on human sperm interphase nucleus. Results ： The expansion of the human sperm was in plain view and the membrane of human sperm cell was integral when it was seen under the inverted microscope ECLIPSE TS100 - F. It was seen under the fluorescence microscope that interphase nucleus counter - stained with DAPI showed blue. The hybridization signal was clear. Conclusions ： This method is effective and could be used for the treatment of human sperm nucleus before FISH.