Quantitative Radioimmunohistochemical Method to Measure the Tissue Content of Specific Molecules

定量放射免疫组织化学方法测定特定分子的组织含量

• 批准号: 02670156
• 负责人: SHIGEMATSU Kazuto
• 金额: $ 1.34万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02670156/
• 关键词: Quantitative Radioimmunohistochemical Method; Immunohistochemistry; Endothelin; Brain Ischemia; Auoradiography; Computerized Densitometry; 定量的radioimmunochemistry法; radioimmunochemistry; オ-トラジオグラフィ-法

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 … More 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

放射免疫组织化学方法与光免疫组织化学方法一样，是识别特定分子定位和分布的有力技术之一。放射自显影已成为显示神经递质受体的常规技术。此外，放射自显影的一个主要优点是可以通过与适当的标准进行比较来进行定量评估。因此，放射自显影技术在放射免疫组织化学中的应用被认为可以定量组织切片中的抗原。在此期间，我们试图建立一种放射组织化学方法来定量测定短暂性前脑缺血后脑组织中内皮素-1(ET-1)的含量。在用冰冷的磷酸盐缓冲液进行腔内灌注后，冰冻的冠状脑切片在细胞停滞液中切开，并解冻安装在覆盖有明胶的玻片上。(A)放射免疫组织化学程序；为了确定实验条件，本研究评估了…研究了ET-1抗血清浓度在1：200~1：4000稀释范围内的影响。(C)孵育温度分别为4ﾟC、22ﾟC或37ﾟC。(B)放射自显影的定量；(A)放射自显影的光学密度(0D)用计算机显微密度法测量。(B)根据与通过加工一整套标准产生的标准曲线的比较，使用0D和蛋白A的比活性来计算^&lt；125；i-蛋白A的量(已知的^&lt；125&>i的浓度增加量)。与切片特异性结合的蛋白A的量与合成的ET-1在硝酸纤维素膜上的量呈线性相关(R=0.98934，P&lt；0.001)。利用此相关曲线，测量组织切片中ET-1的水平。大鼠海马额顶叶皮质FT-1含量为3-7pg/mg，与文献报道的三维酶免疫测定值相近。较少

项目成果

山下 樹三裕: "一過性脳虚血負荷によるエンドセリン産生に関する免疫組織化学的検索" 神経化学. 30. 320-321 (1991)

Kizo Yamashita：“免疫组织化学研究短暂性脑缺血应激诱导的内皮素产生”《神经化学》30. 320-321 (1991)。

Kimihiro Yamashita: "Increased Production of Endothelin Following Tansient Forebrain Ischemia" Bull. Jap. Neurochem.30. 320-321 (1991)

Kimihiro Yamashita：“瞬时前脑缺血后内皮素的产生增加”公牛。