Phalloidin-eosin followed by photo-oxidation: A novel method for localizing F-actin at the light and electron microscopic levels

We describe a novel high-resolution method to detect F-actin at the light a nd electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidatio n of diaminobenzidine. This method possesses several key advantages over an tibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up t o 1%) in the primary fixative, resulting in good ultrastructural preservati on. Second, because both eosin and phalloidin are relatively small molecule s, considerable penetration of reagents into aldehyde-fixed tissue was obta ined 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 f ilaments during preparation for electron microscopy, we were able to visual ize individual actin filaments in some structures. Finally, we show that fl uorescent phalloidin can be directly injected into neurons to label actin-r ich structures such as dendritic spines. These results suggest that the flu orescent phalloidin is an excellent tool for the study of actin networks at high resolution.