Capani F, Deerinck T J, Ellisman M H, Bushong E, Bobik M, Martone M E
Department of Neuroscience, National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, California 92093-0608, USA.
J Histochem Cytochem. 2001 Nov;49(11):1351-61. doi: 10.1177/002215540104901103.
We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.
我们描述了一种新颖的高分辨率方法，可通过使用与荧光团曙红偶联的肌动蛋白结合蛋白鬼笔环肽，随后对二氨基联苯胺进行光氧化，在光学和电子显微镜水平检测F-肌动蛋白。与基于抗体的标记和结构方法相比，该方法具有几个关键优势。首先，鬼笔环肽与F-肌动蛋白的结合能够耐受初次固定剂中相对较高浓度的戊二醛（高达1%），从而实现良好的超微结构保存。其次，由于曙红和鬼笔环肽都是相对较小的分子，无需任何通透步骤，试剂就能大量渗透到醛固定的组织中，从而在电子显微镜水平进行三维重建。通过采用单宁酸二次固定结合低pH值锇酸固定（已知在电子显微镜制备过程中可稳定肌动蛋白丝的条件），我们能够在某些结构中观察到单个肌动蛋白丝。最后，我们表明荧光鬼笔环肽可直接注射到神经元中，以标记富含肌动蛋白的结构，如树突棘。这些结果表明，荧光鬼笔环肽是高分辨率研究肌动蛋白网络的优秀工具。
