Quantitative Radioimmunohistochemical Method to Measure the Tissue Content of Specific Molecules

放射免疫组织化学方法和光免疫组织化学方法是一种有效的识别特定分子定位和分布的技术。放射自显影已成为一种常规的神经递质受体显像技术。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，在放射免疫组织化学中使用放射自显影被认为可以定量组织切片中的抗原。在此，我们试图建立一种放射组织化学方法来定量短暂性前脑缺血后脑组织中内皮素-1（ET-1）的含量。在脑内灌注冰冷的磷酸盐缓冲生理盐水后，冷冻的冠状脑切片在细胞抑制中切开，并解冻安装在明胶包被的载玻片上。(A)放射免疫组化程序；为了确定实验条件，本研究评估了以下参数对ET-1抗血清浓度的影响：(a)研究了ET-1抗血清浓度在初始浓度的1∶200至1∶4000稀释范围内的影响。(b)孵育时间为10 min ~ 48 h。(c)孵育温度为4ºc、22ºc或37ºc。(d) ^<125> I-protein A二抗的浓度从1:10 00开始到1:10 00。(B)放射自成像定量；(a)用计算机微密度法测量自射线摄影的光密度（0D）。(b)通过与处理^<125> - I标准集（已知的^<125> - I浓度增加量）产生的标准曲线进行比较，利用^<125> - I蛋白A的0D和比活性计算^<125> - I蛋白A的量。特异结合到切片上的i -蛋白A的数量呈线性相关（R= 0）。98934， P<0.001)，与合成ET-1在硝化纤维素上的斑点量有关。利用该相关曲线测量组织切片中ET-1的水平。海马至额顶叶皮层FT-1含量范围为3 ~ 7 pg/mg，与三明治酶免疫测定结果相近。少

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