Dual use of immunohistochemistry for film densitometry and light microscopy.
Dual use of immunohistochemistry for film densitometry and light microscopy.
复制标题
双重使用免疫组织化学进行胶片光密度测定和光学显微镜检查。
DOI：
10.1016/j.jneumeth.2012.05.003
复制
发表时间：
2012
影响因子：
3
通讯作者：
Perez-Costas,Emma
中科院分区：
文献类型：
--
作者：
Melendez-Ferro,Miguel;Rice,MatthewW;Roberts,RosalindaC;Perez-Costas,Emma
在本研究中，我们应用免疫印迹和光学显微镜免疫组织化学的原理开发了一种组合方法，可以使用相同的脑组织切片获得胶片中的光密度数据以及载玻片上的形态和蛋白质分布数据，从而最大化从单个样本中获得的数据。当使用非常有价值或独特的组织样本进行实验时，这一点尤其重要，这是人脑研究中非常常见的情况。理想的方法应该结合测量标记物表达水平的可能性以及准确绘制感兴趣区域中该标记物分布的能力。为了实现这一目标，需要满足两件事：首先，该技术需要足够灵敏，以获得标记的光密度或强度测量，其次，需要良好保存组织以研究分布模式和形态分析。在这里，我们表明，我们的组合方法对于不同的组织保存条件（新鲜冷冻和固定的组织）、不同的物种（大鼠和人类）、不同的大脑区域（黑质和纹状体）以及不同标记物（酪氨酸羟化酶和μ-阿片受体）的检测产生了可靠的结果。该方法还将胶片中光密度数据采集的准确性与从同一样本获取组织学切片相结合。总之，这里提出的方法非常通用，除了解剖实验室中存在的常规设备之外，不需要使用专门的设备。
In the present study, we applied the principles of immunoblotting and light microscopy immunohistochemistry to develop a combined methodology that allows obtaining optical density data in films, as well as morphological and protein distribution data on slides using the same brain tissue section, thus maximizing the data obtained from a single sample. This is especially important when experiments are performed using very valuable or unique tissue samples, which is a very common case in the study of the human brain. The ideal methodology should combine the possibility of measuring levels of expression of a marker, and the capability to map accurately the distribution of that marker in the region of interest. To achieve this, two things are required: first, the technique needs to be sensitive enough to obtain optical density or intensity measurements of the marker, and second, a good preservation of the tissue is needed for the study of distribution patterns and morphological analysis. Here we show that our combined methodology produced reliable results for different tissue preservation conditions (fresh-frozen and fixed tissue), in different species (rat and human), in different brain areas (substantia nigra and striatum), and for the detection of different markers (tyrosine hydroxylase and μ-opioid receptor). This methodology also combines the accuracy of optical density data acquisition in film with obtaining histological slides from the same sample. In summary, the methodology proposed here is very versatile and does not require the use of specialized equipment, other than the routine equipment present in an anatomy laboratory.