Optimal Conditions for Detection of Calcitonin mRNA by In Situ Hybridization (ISH) Method Using a Non-radioactive Probe in the Rat Thyroid Gland
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- NAKAJIMA Takayuki
- <I>Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University</I>
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- OGAWA Kazushige
- <I>Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University</I>
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- TANIGUCHI Kazuyuki
- <I>Department of Veterinary Anatomy, Faculty of Agriculture, Iwate University</I>
Bibliographic Information
- Other Title
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- 非放射性プローブを用いたin situ hybridization (ISH) 法によるカルシトニンmRNA検出の最適条件の検討
- ヒ ホウシャセイ プローブ オ モチイタ in situ hybridizat
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Abstract
Optimal conditions for detection of calcitonin mRNA were examined by in situ hybridization (ISH) method using a non-radioactive probe in the rat thyroid gland. An oligonucleotide complementary to rat calcitonin mRNA was synthesized with a DNA synthesizer, labelled at 3'-end by using digoxigenin-11-deoxyuridine triphosphate (dUTP) and terminal transferase (Boehringer Mannheim), and used as a probe. Hybrid chains formed by probe and calcitonin mRNA were visualized by anti-digoxigenin-alkaline phosphatase conjugate, nitroblue tetrazolium (NBT) and X-phosphate (Boehringer Mannheim) . To determine the optimal conditions for ISH, relations between tissue fixation and proteinase K treatment, and between hybridization temperature and time were mainly surveyed in the present study. As for the relation between tissue fixation and proteinase K treatment, good results were obtained in sections fixed by immersion in 10% formalin at 4°C for 2 hr, and digested with 1 μg/ml proteinase K at 37°C for 20 min. Under this condition, the most intense signals were obtained after hybridization at 37°C overnight. Alternative application of ISH and immunostaining to each of the adjacent sections revealed a small number of cells which were immunonegative but displayed hybridization signals.
Journal
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- Experimental Animals
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Experimental Animals 43 (4), 477-486, 1994
Japanese Association for Laboratory Animal Science
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Details 詳細情報について
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- CRID
- 1390001205501635584
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- NII Article ID
- 130005001160
- 130006135317
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- NII Book ID
- AN00104954
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- ISSN
- 18817122
- 00075124
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- NDL BIB ID
- 3910271
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- Data Source
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- JaLC
- NDL
- Crossref
- CiNii Articles
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- Abstract License Flag
- Disallowed