Schroeder-Reiter E, Houben A, Wanner G
Department of Biology I, Ludwig-Maximillians-Universität München, Menninger Strasse 67, D-80638 Munich, Germany.
Chromosome Res. 2003;11(6):585-96. doi: 10.1023/a:1024952801846.
High-resolution detection of phosphorylated histone H3 at serine 10 in mitotic barley chromosomes for scanning electron microscopy was shown using a novel application of indirect immunogold labeling with Nanogold. This method permits localization and quantification of signals in a three-dimensional context. Because the chromosome structure is well preserved, characterization of binding sites (chromomeres, parallel matrix fibers, solenoids), currently in the realm of nanometer decades, is possible. Quantification and three-dimensional localization of labels is possible with stereoscopic analysis. Limitations of the method pertain to the challenges in preservation of chromosome ultrastructure, accessibility of immunoreactants into the fixed chromatin and unspecific labeling. The differences between silver and gold enhancement and the current status of labeling efficiency are addressed.
利用纳米金间接免疫金标记的新应用，展示了用于扫描电子显微镜的有丝分裂大麦染色体中丝氨酸10位点磷酸化组蛋白H3的高分辨率检测。该方法允许在三维环境中对信号进行定位和定量。由于染色体结构保存良好，目前处于纳米级别的结合位点（染色粒、平行基质纤维、螺线管）的表征成为可能。通过立体分析可以对标记进行定量和三维定位。该方法的局限性涉及染色体超微结构保存方面的挑战、免疫反应物进入固定染色质的可及性以及非特异性标记。文中还讨论了银增强和金增强之间的差异以及标记效率的当前状况。
