Quantitative Radioimmunohistochemical Method to Measure the Tissue Content of Specific Molecules

放射免疫组化法与光免疫组化法一样，是鉴定特异性分子定位和分布的有效方法之一。放射自显影已成为神经递质受体显像的常规技术。此外，放射自显影的一个主要优点是通过与适当的标准进行比较来进行定量评估的可能性。因此，在放射免疫组化中使用放射自显影被认为可以定量组织切片中的抗原。本研究旨在建立一种放射组织化学方法，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盐水进行颅内灌注后，将冷冻的冠状脑切片在细胞停滞中切割并解冻固定在明胶包被的载玻片上。(A)放射免疫化学程序;为了确定实验条件，本研究评估了 ...更多信息 以下参数的影响：（a）研究ET-1抗血清的浓度，从初始浓度的1：200到1：4000稀释。（B）孵育时间为10 min ~ 48 h。（c）孵育温度为4 ℃、22 ℃或37 ℃。（d）<125>从1：1000至1：100开始β 1-蛋白A二抗的浓度。（B）放射自显影图的定量;（a）通过计算机化显微密度测定法测量放射自显影图的光密度（OD）。（B）<125>使用OD和^I-蛋白A的比活性计算^I-蛋白A的量<125>，基于与通过处理^<125>I-标准品组（已知量的递增浓度的^<125>I）生成的标准曲线的比较。与<125>切片特异性结合的α-I-蛋白A的量呈线性相关（R= 0. 98934，P&lt;0.001），其中合成的ET-1的量点在硝酸纤维素上。使用该相关曲线，测量组织切片中的ET-1水平。海马至额顶叶皮质FT-1含量范围为3-7 pg/mg，与用酶联免疫法测定的结果相近。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