ESTABLISHMENT OF IN SITU HYBRIDIZATION AT ELECTRON MICROSCOPIC LEBEL

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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.