Eizuru Y, Minamishima Y, Matsumoto T, Hamakado T, Mizukoshi M, Nabeshima K, Koono M, Yoshida A, Yoshida H, Kikuchi M
Department of Microbiology, Miyazaki Medical College, Japan.
J Virol Methods. 1995 Apr;52(3):309-16. doi: 10.1016/0166-0934(94)00163-b.
Non-isotopic in situ hybridization with a novel phenytoin (PHE)-labeled probe was developed. The mixture of cloned cytomegalovirus (CMV) DNA fragments was labeled by random primer technique using PHE-11(spacer)-dUTP, instead of dTTP. The tissue sections were treated with 0.2 N HCl and with proteinase K (1 microgram/ml), and then heated at 70 degrees C in the presence of 50 or 75% formamide. The sections were hybridized with PHE-labeled probe at 37 degrees C overnight. The hybridization signal was visualized by alkaline phosphatase-5-bromo-4-chloro-3-indolyl phosphate (BCIP)/4-nitroblue tetazolium (NBT) system. Strong hybridization signals were detected in sections of the small intestine and the placenta, even when denatured in the presence of 50% formamide. In the case of small intestine, CMV DNA was also detected in the endothelial cells of the mucosa where apparent infected cell was not observed histologically. In the sections of the submaxillary gland, the lung, the adrenal gland and the ovary, hybridization signal was not detected when denatured in the presence of 50% formamide, but detected after denaturation with 75% formamide. Thus, in situ hybridization with the novel PHE-labeled probe is applicable to conventional formalin-fixed, paraffin-embedded tissue sections.
开发了一种用新型苯妥英（PHE）标记探针的非同位素原位杂交技术。使用PHE-11（间隔物）-dUTP而非dTTP，通过随机引物技术对克隆的巨细胞病毒（CMV）DNA片段混合物进行标记。组织切片先用0.2N盐酸和蛋白酶K（1微克/毫升）处理，然后在50%或75%甲酰胺存在的情况下于70℃加热。切片在37℃与PHE标记的探针杂交过夜。通过碱性磷酸酶-5-溴-4-氯-3-吲哚磷酸（BCIP）/4-硝基蓝四唑（NBT）系统观察杂交信号。即使在50%甲酰胺存在的情况下变性，在小肠和胎盘切片中仍检测到强杂交信号。在小肠中，在组织学上未观察到明显感染细胞的黏膜内皮细胞中也检测到了CMV DNA。在颌下腺、肺、肾上腺和卵巢的切片中，在50%甲酰胺存在的情况下变性时未检测到杂交信号，但在75%甲酰胺变性后检测到了。因此，用新型PHE标记探针进行的原位杂交适用于常规福尔马林固定、石蜡包埋的组织切片。
