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ESTABLISHMENT OF IN SITU HYBRIDIZATION AT ELECTRON MICROSCOPIC LEBEL

电子显微镜LEBEL原位杂交的建立

基本信息

批准号：
06557017
负责人：
MAEDA Sakan
金额：
$ 3.33万
依托单位：
KOBE UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

In situ hybridization (ISH) at electron microscopic level using bromodeoxyuridine (BrdU) labelled DNA probe was tried to establish and several conditions were examined to conserve the precise ultrastructure for the recognition of hybrid location. BrdU labeled probes were obtained by the addition of BrdU in medium and used for ISH at electron microscope. Shortly, cell were hybridized with BrdU labeled probe prior to embedding. Then cells were covered with Quetol block and embedded. The post-embedding immunoelectron microsopic method using anti-BrdU monoclonal antibody was performed on the thin section. Then the sections were observed by electron microscope.Tissue fixation was important step to obtain the original ultrastructure and tried to use microwave (MI77 type MICROWAVE PROCESSOR,AZUMAYA,TOKYO). In the antigen retrieval immunostaining, p53 antigenecity recovered even the samples stored in un-buffered formalin for several months. However the difference between the microwave treated and un-treated groups in IHS at electron microscopic level was not clear.On January 17,1995 Kobe was attacked by the Hanshin-Awaji great earthquake and our electron microscope was severely damaged. Nine months later the electron microscope was repaired and our study continued.This methods were appied for polymerase chain reaction (PCR)-ISH and the detection of alternative splicing. PCR-ISH was difficult because of the movement of amplified DNA.The alternative splicing was observed at cytoplasm by the use of several combination of exons.Further study is necessary to establish the ISH at electron microscope.

用溴脱氧尿嘧啶核苷(BrdU)标记的DNA探针在电子显微镜水平上进行原位杂交(ISH)，并对几种条件进行了考察，以保存杂交定位所需的精确超微结构。通过在介质中加入BrdU得到BrdU标记的探针，并用于电子显微镜下的ISH。不久，细胞与BrdU标记的探针杂交后，再进行包埋。细胞包被奎托封闭剂包埋。用抗BrdU单抗进行包埋后免疫电子显微镜检查。组织固定是获得原始超微结构的重要步骤，并尝试使用微波(东京Azumaya的MI77型微波处理机)。在抗原修复免疫染色中，即使在无缓冲福尔马林中保存几个月的标本，P53抗原性也能恢复。然而，微波处理组和未处理组的IHS在电子显微镜水平上的差异并不明显。1995年1月17日，神户遭受阪神-Awaji大地震的袭击，我们的电子显微镜严重受损。9个月后，电子显微镜被修复，我们的研究继续进行。这些方法被应用于聚合酶链式反应(PCR)-ISH和选择性剪接的检测。由于扩增DNA的移动，PCR-ISH很困难，通过几个外显子的组合在细胞质上观察到选择性剪接，在电子显微镜下建立ISH还需要进一步的研究。