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首页> 外文期刊>Brain research. Brain research protocols >Comparison of contrast, sensitivity and efficiency of signal amplified and nonamplified immunohistochemical reactions suitable for videomicroscopy-based quantification and neuroimaging.
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Comparison of contrast, sensitivity and efficiency of signal amplified and nonamplified immunohistochemical reactions suitable for videomicroscopy-based quantification and neuroimaging.

机译：比较适用于基于视频显微镜的定量和神经成像的信号放大和非放大的免疫组织化学反应的对比度，灵敏度和效率。

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In recent years, many different technical modifications of immunohistochemical methods have been developed. The selection of a suitable technique for quantitative purposes such as mapping studies can be quite difficult. Various features of a certain method must be considered such as the sensitivity, costs, duration and practicability with respect to serial sectioned specimens. Background and foreground difference or contrast and the influence of artifacts are major problems of quantitative immunohistochemistry. It is not known which of the different modifications of immunohistochemical signal amplifications and non-amplifications gives optimal results in respect to image analytical-based quantification. However, for image analysis, it is important to analyze sections which offer a sufficient contrast between foreground and background. The sensitivity of a system is crucial when quantitative immunohistochemistry should be applied to scarce material with longer postmortem and storage times which occur often by processing human brains. In addition, the enzyme-substrate reactions have an obvious influence on this criterion; therefore, different substrates were also tested. The contrast may be as well effected by the quality and specificity of the primary antibody, the type of tissue and naturally by preparative (fixation, postmortem delay, storage) and individual factors (age, circadian effects, diseases, sex). Because all of these factors may yield to different results by combining them with different neuronal structures, we used three different antigen expressions for a specific analysis: fibrillary, granulary and perikaryal antigen distributions in brains from Wistar rats. Principally, the sensitivity of the modifications of immunohistochemical amplifications is revealed more strongly than without enhancement steps; however, the contrast between foreground and background structures does not necessary increase by applying a certain amplification technique. The lowest contrast (15%) was detected after applying the labelled streptavidin-biotin technique. All other methods offer comparable contrasts in between 30% and 40%. The catalyzed signal amplification reaction has been found to give optimal results (40% contrast) for image analysis. However, from the technical point of view and variability of protein expression, storage and postmortem delay, it was necessary to adapt the commercial CSA Kit from Dako (K1500). The modified technique, called C2 method, offers better results with respect to sensitivity, total costs, duration and contrast (60%) and variability of contrast.
机译：近年来，已经开发了免疫组织化学方法的许多不同的技术修饰。为定量目的（例如作图研究）选择合适的技术可能非常困难。必须考虑某种方法的各种特征，例如对于连续切片的标本的敏感性，成本，持续时间和实用性。背景和前景的差异或对比度以及伪影的影响是定量免疫组织化学的主要问题。关于基于图像分析的定量，免疫组织化学信号放大和非放大的不同修饰中的哪一种给出了最佳结果，这是未知的。但是，对于图像分析，重要的是分析在前景和背景之间具有足够对比度的部分。当定量免疫组织化学应应用于具有较长死后和储存时间的稀有材料时，系统的灵敏度至关重要，而稀有材料的储存和保存时间通常是通过加工人脑而发生的。另外，酶-底物反应对该标准有明显影响。因此，还测试了不同的基材。对比也可能受一抗的质量和特异性，组织类型的影响，自然也受制备性（固定，死后延迟，储存）和个体因素（年龄，昼夜节律，疾病，性别）的影响。由于所有这些因素都可能通过将它们与不同的神经元结构组合在一起而产生不同的结果，因此我们使用三种不同的抗原表达进行特定分析：Wistar大鼠大脑中的纤维状，颗粒状和周围核抗原分布。原则上，与没有增强步骤相比，更强烈地揭示了免疫组织化学扩增修饰的敏感性。然而，前景和背景结构之间的对比度不必通过应用某种放大技术来增加。应用标记的抗生蛋白链菌素-生物素技术后，检测到最低的对比度（15％）。所有其他方法在30％到40％之间提供可比的对比度。已发现催化信号放大反应可为图像分析提供最佳结果（对比度为40％）。但是，从技术角度以及蛋白质表达，储存和验尸延迟的可变性来看，有必要对Dako（K1500）的商业CSA试剂盒进行改造。改进的技术称为C2方法，在灵敏度，总成本，持续时间和对比度（60％）和对比度可变性方面提供了更好的结果。

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