【摘要】 以Biotin标记的dATP(Bio-7-dATP)通过缺刻翻译(Nick Translation)制备腺病毒的基因组探针,建立了电镜原位杂交技术,并研究腺病毒感染的HeLa细胞内病毒DNA的分布。结果表明,免疫胶体金颗粒特异地标记在腺病毒感染的细胞核内,并且在核内呈区域性分布。整装抽提后的细胞原位杂交结果显示出,胶体金颗粒呈族状马串珠状结合在核骨架上,从而在形态学上证实了腺病毒DNA与核骨架的紧密结合关系。更多还原

【Abstract】 Biotinylated dATP (Bio-7-dATP) and 3H-dTTP labeled adenovirus DNA were hybridized in situ to thin sec-tions of Lowicryl K4M-embedded and whole-mount extracted HeLa cells infected with adenovirus. The biotinatedprobe was detected by exposing the cell or section to antibodies against biotin followed by colloidal gold-conjugatedsecondary antibodies and then criticalpoint dried while ’~H-dTTP labeled probe by electron microscopic au-toradiography. On Lowicryl K~M sections, gold particles and silver grains were mainly restricted in the nucleus.Furthermore, whole-mount results suggested that replicating adenovirus DNA is localized on the nuclear matrix ofits host cell. In this paper, the described nonradioactive procedures for hybrid detection offered several advantages:(a) rapid signal detection; (b) superior morphological preservation and spatial resolution; (e) precise localization;and (d) on Lowicryl K4M sections, signal to noise equivalent to radiolabeling.更多还原