ESTABLISHMENT OF IN SITU HYBRIDIZATION AT ELECTRON MICROSCOPIC LEBEL

用溴脱氧尿苷（BrdU）标记的DNA探针，在电镜水平上建立了原位杂交（ISH）技术，并考察了几种条件，以保存精确的超微结构，用于杂交定位的识别。在培养液中加入BrdU，获得BrdU标记探针，用于电镜下的原位杂交。在包埋前，细胞与BrdU标记的探针杂交。然后用Quetol块覆盖细胞并包埋。采用抗BrdU单克隆抗体包埋后免疫电镜方法。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（MI 77型微波仪，AZUMAYA，TOKYO）。在抗原修复免疫染色中，即使在非缓冲福尔马林中储存数月的样品也能恢复p53抗原性。1995年1月17日，科比发生坂神淡路大地震，我们的电镜遭到严重破坏。9个月后，电镜修复后继续进行研究，该方法适用于聚合酶链反应（PCR）-ISH和选择性剪接的检测。PCR-ISH因扩增DNA的移动而困难，但在细胞质中观察到选择性剪接，采用多种外显子组合，电镜下ISH的建立还需进一步研究。

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.