| Project/Area Number |
03557001
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Shiga University of Medical Science(SUMS) |
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Principal Investigator |
KIMURA Hiroshi SUMS, Molecular Neurobiology Research Center, Professor., 分子神経生物学研究センター, 教授 (40079736)
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| Co-Investigator(Kenkyū-buntansha) |
TOOYAMA Ikuo SUMS, Molecular Neurobiology Research Center, Assistant, 分子神経生物学研究センター, 助手 (20207533)
中村 伸 島津製作所, 技術課, 研究員
柏木 克也 島津製作所, 技術課, 研究員
大杉 義彰 島津製作所, 技術課, 主任
秋山 純一 島津製作所, 技術部, 副部長
神谷 鉄也 東亜合成化学工業, 研究員
吉田 祇生 東亜合成化学工業, 主任研究員
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥15,600,000 (Direct Cost: ¥15,600,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥14,800,000 (Direct Cost: ¥14,800,000)
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| Keywords | biotin phosphoramidite / vasopressin / quantitative image analysis / in situ hybridization / electron microscopy / probe design / プロ-ブ / in situハイブリダイゼ-ション |
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| Research Abstract |
High resolution non-radioactive in situ hybridization histochemistry using chemically defined probes has been developed and applied for the detection of vasopressin mRNA in the paraventricular and supraoptic nucleus of the rat. Defined number of biotins were directly incorporated at specified positions in the probe nucleotide sequence with a DNA synthesizer using a biotin phosphoramidite reagent. Hybridization signals obtained by different probes based on different desings (number and positions of biotins, spacer arm length) were quantitatively examined using an image analyzer. The staining intensity was significantly higher with a prove having 5 biotins at 3' terminal than probe having 2 biotins (p<0.001). Although a probe having 10 biotins gave a further high staining intensity than the probe having 5 biotins, the increment was rather smaller (approximately 12%) and not significant than expected. Little difference was observed with 5' and 3' terminally labeled probes of the same extent of biotinylation. Poor signals were observed with the probes of 20 biotins or 5 biotins within the sequence. The results suggest that probes labeled with 5 biotins at either 5' or 3' terminal are suitable for in situ hybridization histochemistry. With this staining technique, in combination of peroxidase/ diaminobenzidinne visualizing reaction, it is possible not only to quantify histochemical results but also to observe the intracellular localization of vasopressin expression at an electron microscopic level.
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