Abstract：
Correlative microscopy is an important approach for bridging the resolution gap between fluorescence light and electron microscopy. Here, we describe a fast and simple method for correlative immunofluorescence and immunogold labeling on the same section to elucidate the localization of phosphorylated vimentin (P-Vim), a robust feature of pulmonary vascular remodeling in cells of human lung small arteries. The lung is a complex, soft and difficult tissue to prepare for transmission electron microscopy (TEM). Detailing the molecular composition of small pulmonary arteries (<500μm) would be of great significance for research and diagnostics. Using the classical methods of immunochemistry (either hydrophilic resin or thin cryosections), is difficult to locate small arteries for analysis by TEM. To address this problem and to observe the same structures by both light and electron microscopy, correlative microscopy is a reliable approach. Immunofluorescence enables us to know the distribution of P-Vim in cells but does not provide ultrastructural detail on its localization. Labeled structures selected by fluorescence microscope can be identified and further analyzed by TEM at high resolution. With our method, the morphology of the arteries is well preserved, enabling the localization of P-Vim inside pulmonary endothelial cells. By applying this approach, fluorescent signals can be directly correlated to the corresponding subcellular structures in areas of interest.
摘要：
相关显微镜是桥接荧光和电子显微镜之间分辨率差距的重要途径。这里,我们描述了一个快速和简单的方法相关的免疫荧光和免疫金标记在同一部分阐明磷酸化波形蛋白（P-Vim）的定位，人肺小动脉细胞肺血管重塑的强大特征。肺是一个复杂的，柔软和困难的组织准备透射电子显微镜（TEM）。详细说明小肺动脉（<500μm）的分子组成对于研究和诊断具有重要意义。使用经典的免疫化学方法（亲水树脂或薄冷冻切片），很难定位小动脉进行透射电镜分析。为了解决这个问题，并通过光学和电子显微镜观察相同的结构，相关显微镜是一种可靠的方法。免疫荧光使我们能够知道P-Vim在细胞中的分布，但不能提供其定位的超微结构细节。通过荧光显微镜选择的标记结构可以通过TEM以高分辨率进行鉴定和进一步分析。用我们的方法,动脉的形态保存完好,使P-Vim在肺内皮细胞内定位。通过应用这种方法，荧光信号可以与感兴趣区域中的相应亚细胞结构直接相关。
