DETECTION OF ENZYME ACTIVITIES ON ULTRATHIN FROZEN SECTIONS - A NEW METHOD FOR ELECTRON MICROSCOPIC ENZYME HISTOCHEMISTRY.

超薄冰冻切片上酶活性的检测——电子显微镜酶组织化学的新方法。

• 批准号: 05670030
• 负责人: FUKUSHIMA Osamu
• 金额: $ 1.41万
• 依托单位: JIKEI UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670030/
• 关键词: ULTRATHIN FROZEN SECTION; ENZYME HISTOCHEMISTRY; ACID PHOSPHATASE; ALKALINE PHOSPHATASE; AMINOPEPTIDASE; DIPEPTIDYL PEPTIDASE; CYTOCHROME C OXIDASE; cytochrome Coxidase; 凍結超薄切片; cytochrome C oxidase; 蛋白分解酵素; cytochrome c oxidase

The aim of electron microscopic enzyme histochemistry is to localize the sites of enzyme activities on cellular ultrastructure on order to understand the metabolism of cells. The routine enzyme histochemical techniques employed 40 um sections for demonstrating activities. Our study shows that there are penetration problems in 40 um sections for detection of enzyme activities, especially cytochrome C oxidase activity, and concluded that the ultrathin frozen section is suitable for demonstrating it at electron microscopic level. The study indicates that a histochemical reaction should be done on an ultrathin section. There are two methods for histochemical reactions ; one is an ultrathin frozen section, and the other a plastic ultrathin section. A plastic embedding needs a dehydration procedure. However, our study indicates the displacement of enzyme ptoteins during a dehydration procedure. The ultrathin frozen sections had not been exposed to organic solvents prior to demonstration of enzyme activities. The study shows that an ultrathin frozen section is most suitable for demonstrating enzyme sctivities directly on an ultrathin section. The present study concludes that an ultrathin frozen section should be employed for observation of precise localization of an enzyme activity at electron microscopic level.

电镜酶组织化学的目的是定位酶在细胞超微结构上的活性部位，以了解细胞的代谢。常规酶组织化学技术采用40 μ m切片显示活性。我们的研究表明，在40 μ m切片中检测酶活性，特别是细胞色素C氧化酶活性存在穿透问题，并得出结论，冷冻切片适合于在电子显微镜水平上显示。研究表明，组织化学反应应在组织切片上进行。组织化学反应有两种方法，一种是冰冻切片，另一种是塑料切片。塑料包埋需要脱水程序。然而，我们的研究表明，在脱水过程中酶蛋白的位移。在证明酶活性之前，未将冷冻切片暴露于有机溶剂。研究表明，直接在冰冻切片上显示酶的活性，最适宜的是冰冻切片。本研究的结论是，在电镜水平上观察酶活性的精确定位，应采用快速冰冻切片。

项目成果

福島統: "An ultrathin frozen section is suitable for demonstrating cytochrome C oxidase activity at electron microscopic level." Journal of Electron Microscopy. 42. 351-355 (1993)

Osamu Fukushima：“超薄冷冻切片适合在电子显微镜水平上展示细胞色素 C 氧化酶活性。”《电子显微镜杂志》42. 351-355 (1993)。

Fukushima O: "Crto-techniques for the enzyme histochemistry (in Japanese)." Saibou. 26. 512-515 (1994)

Fukushima O：“酶组织化学的 Crto 技术（日语）”。

Fukushima O.: "Observation of intracellular large molecules with cryo-techniques (in Japanese)." Soshikisaibou. 20. 356-368 (1994)

Fukushima O.：“用冷冻技术观察细胞内大分子（日语）”。

福島統: "凍結超薄切片酵素組織化学" 日本組織細胞化学会編、組織細胞化学1994. 206-212 (1994)

福岛修：《冰冻超薄切片酶组织化学》日本组织细胞化学学会编，组织细胞化学 1994. 206-212 (1994)

福島 統: "An ultrathin frozen section is suitable for demonstrating cytochrome c oxidase activity at electron microscopic level." Journal of Electron Microscopy. 42. 351-355 (1993)

Osamu Fukushima：“超薄冷冻切片适合在电子显微镜水平上展示细胞色素 C 氧化酶活性。”《电子显微镜杂志》42. 351-355 (1993)。