论文部分内容阅读
原位杂交法是用直接组织切片检测特定的遗传基因的方法。近年来,通过遗传基因工程单克隆技术获得了高纯度的探针,用切口翻译法获得了活性高的DNA 探针,故应用范围越来越广。可是,目前的方法,通常是以新鲜冰冻切片作原料,使用3~H 和~(125)I 标记的DNA 探针,靠自动放射照相术检测。本文作者以福尔马林固定的石蜡包埋组织作原料,用生物素标记DNA 探针进行杂交,通过使用过氧化酶的 ABC 法,检测肝组织内 HBVDNA。原料和方法:分别将4μm 厚的2例经福尔马林固定的 HBeAg 阳性肝硬变患者的尸检肝标本石蜡包埋组织薄切片,贴在用 Denhardt 液处理过的载玻片上,脱石蜡后,用 Carnoy 液再固定。然后作以下前处理:1%三硝基甲苯 X100室温2分钟,0.2N HCI 室温2分钟,2XSSC70℃2分钟,1μg/ml 蛋白酶 K 室温15分钟,
In situ hybridization is a method of detecting specific genes using direct histological sections. In recent years, high-purity probes have been obtained by genetic engineering of monoclonal technology, and high-activity DNA probes have been obtained by nick-translation method. Therefore, the application range is wider and wider. However, the current method, usually using fresh frozen sections as raw materials, is detected by automatic radiography using 3 H and 125 I labeled DNA probes. In this study, formalin-fixed paraffin-embedded tissue was used as a starting material, and biotin-labeled DNA probes were used for hybridization. HBVDNA in liver tissues was detected by the ABC method using peroxidase. MATERIALS AND METHODS: Two sections of autopsy liver specimens of formalin-fixed HBeAg-positive cirrhotic patients were embedded in 2 μm thick sections of paraffin-embedded tissue sections on the slides treated with Denhardt’s solution. After deparaffinization , Fix with Carnoy solution. Then the following pretreatment: 1% trinitrotoluene X100 room temperature for 2 minutes, 0.2N HCI room temperature for 2 minutes, 2XSSC70 ° C for 2 minutes, 1μg / ml Proteinase K for 15 minutes at room temperature,