| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Research Abstract |
Radioimmunohistochemical method as well as light immunohistochemical method is one of powerful technique avaibable for identifying the localization and distribution of specific molecules. Autoradiography has become a routine technique for visi, alization of neurotransmitter receptor. Furthermore, a major advantage of autoradiography is the possibility of quantitative assesments by comparison with appropriate standards. Hence, the use of autoradiography in radioimmunohistochemistry is considered to allow quantification of antigens in the tissue sections. In this time, we attempted to develop a radio ohistochemical method to quantify endothelin-1(ET-1)contents in brain tissues after a transient forebrain ischemia. After perfusion intracanadially with ice cold posphate buffered saline, frozen coronalbrain sections were cut in a cytostasis and thaw-mounted onto gelatin-coated slides. (A) Radioimmunohistoshemical procedure ; In order to define experimental conditions, the study assessed the
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effects of the following parameters : (a)the concentration of ET-1 antiserlun was studied from 1 : 200 to 1 : 4000 dilution of the initial concentration. (b)the incubation time was tested from 10 min to 48 h. (c)the temperature for incubation was carried out 4ﾟC, 22ﾟC or 37ﾟC. (d)the concentration of ^<125> I-protein A secondary antibody was started f rom 1 : 1000 to 1 : 100. (B)Quantification of autoradiograms ; (a)the optical densities(0D)of autoradiograms were measured bu computerized microdensitometry. (b)the amount of ^<125>I-protein A was calculated using 0D and specific activity of ^<125>I-protein A, as based on a comparison with standards curves generated by processing sets of ^<125>I-standards (known amounts of increasing concentrations of ^<125>I). The mount of ^<125>I-protein A specifically bound to the sections showed a linear correlation(R=o. 98934, P<0.001)with the amount of synthetic ET-1 spotted onto nitrocellurose. Using this correlation curve, ET-1 levels in the tissue sections were measured. FT-1 contents in the hippocampus to frontoparietal cortex ranged 3-7 pg/mg, the values of which were in a similar range to those reported using sandwichenzyme immunoassay. Less
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