In situ detection of reactive oxygen species and nitric oxide production in normal and pathological tissues: improvement by differential interference contrast
In situ detection of reactive oxygen species and nitric oxide production in normal and pathological tissues: improvement by differential interference contrast
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DOI：
10.1016/s0531-5565(01)00226-1
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发表时间：
2002-04-01
影响因子：
3.9
通讯作者：
Vairetti, M
中科院分区：
文献类型：
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作者：
Freitas, I;Griffini, P;Vairetti, M
目前包括一氧化氮（NO）在内的活性氧（ROS）发挥着多种（病理）生理作用。哈曼40多年前提出的自由基衰老理论从此得到了充分的支持。McCann最近提出的一氧化氮老化假说，很可能在不久的将来成为广泛研究的对象。因此，在光镜下用简单可靠的方法在（亚）细胞水平上定位ROS和NO产生位点的可能性成为细胞学和病理学分析的有力工具。开发了各种组织化学方法来可视化ROS的产生；最近，一种基于DAB-Mn2+-Co2+反应的超氧化物（也可能是单线态氧）定位的改进版本看起来非常有希望。由于直接检测NO仍然非常困难，目前通过鉴定NO合成酶（NOS）来定位NO的作用位点。揭示NOS催化活性的最广泛的方法是用四氮唑盐法证明抗固定的NADPH diaphorase活性。我们改进了这种方法，使用了一种四氮唑盐（四硝基蓝四氮唑，TNBT），这种盐的甲醛颗粒非常薄，脂不溶性很好，并且在培养培养基中加入了一种组织保护剂聚乙烯醇。本文介绍了将DAB-Mn2+-Co2+技术用于ROS和TNBT-PVA方法用于NOS在正常肝、脑和实体瘤中的应用。我们进一步记录了Nomarkis的差分干扰对比（DIC）在分析ROS产生或NOS活性低甚至为零的广泛组织区域的实用性。NOS的改进版本首次证明了NOS在肝脏脂肪储存细胞和大脑星形细胞样细胞中的活性。(C) 2002爱思唯尔科学有限公司版权所有。
Reactive oxygen species (ROS), among which nitric oxide (NO) is currently included, play a plethora of (patho)physiological roles. Harman's free radical theory of aging put forth over 40 years ago received full support since then. A nitric oxide hypothesis of aging recently proposed by McCann, is very likely to be the object of widespread investigation in the near future. Therefore, the possibility of localizing at the (sub)cellular level under the light microscope the sites of ROS and NO production with simple and reliable methods appears as a powerful tool for analytic cytology and pathology. Various histochemical methods were developed for visualizing ROS production; a recently improved version to localize superoxide (and possibly also singlet oxygen), based on a DAB-Mn2+-Co2+ reaction, appears very promising. Since the direct detection of NO is still very difficult, the action sites of NO are currently localized by the identification of NO synthase (NOS). The most widespread method to reveal the catalytic activity of NOS is that of demonstrating the fixation-resistant NADPH diaphorase activity with the tetrazolium salt method. We have improved this method by using a tetrazolium salt whose formazan particles are very thin and lipid insoluble (tetranitroblue tetrazolium, TNBT) and by including a tissue protectant, polyvinyl alcohol, in the incubation medium. Here significant examples of application of the DAB-Mn2+-Co2+ technique for ROS and the TNBT-PVA method for NOS to normal liver and brain and to solid tumors are presented. We further document the usefulness of Nomarkis's differential interference contrast (DIC) to analyze wide tissue areas where ROS production or NOS activity is low or even nil. The improved version for NOS allowed for the first time to demonstrate NOS activity in liver fat-storing cells and in astrocyte-like cells in the brain. (C) 2002 Elsevier Science Inc. All rights reserved.