| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 1985: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1984: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
During the past two decades, electron-immunohistochemical methods have been improved and are indispensable tool for the pathological diagnosis. However those methods are cumbersome and require specialized instruments. Consequently, they have not been used in routine pathology laboratories. In order to make them practical, we investigated the possibility of observing the light microscopic preparations by backscattered electron image (BEI) formed by scanning electron microscope (SEM). JEOL T200 SEM was modified for this purpose. For the observation by BEI, the tissue sections on light microscopic slide must be grounded, since the slide insulate the electrons and result in electron charging at the point of electron impact. Among various organic and inorganic materials examined, ferric ammonium sulfate (FAS) was found to be most practical for the prevention of electron charging. To gain contrast at the sites of antigens, the tissue sections require staining with heavy metals. We found that a repeated exposure to osmium and thiocarbohydrazide was best suited for this purpose. A procedure for the localization of antigen by BEI is summarized;
A. One to 2 microns section of fixed tissues embedded in synthetic resin is placed on glass slide and is reacted with horseradish peroxidase conjugated antibody. The peroxidase is then localized using diaminobenzidine and H202.
B. The reaction products is chelated with first with osmium, then with thiocarbohydrazide and again with osmium. The section is finally counter-stained with uranyl acetate and treated with FAS.
Although with the current instrument, the resolution of the BEI was not as good as that of transmission electron microscope, we were able to localize antigens with the above method with a resolution which is unattainable with the ordinary light microscope. We expect the resolution should be improved as more modifications are made on the instrument and with the method of staining.
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