Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling

Abstract

Recently, three-dimensional reconstruction of ultrastructure of rat brain has been realized with minimal effort by using scanning electron microscopy combined with focused ion beam milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy provides a great advantage in that molecules of interest are specifically localized in ultrastructures. Thus, we applied immunocytochemistry for FIB-SEM and correlated this immunoreactivity with that in confocal laser-scanning microcopy (CF-LSM). Dendrites of medium-sized spiny neurons in the rat neostriatum were visualized using a recombinant viral vector, which labeled the infected neurons with membrane-targeted GFP in a Golgi stain-like fashion. Moreover, the thalamostriatal afferent terminals were immunolabeled with Cy5 fluorescence for vesicular glutamate transporter 2 (VGluT2). After detection of the sites of terminals apposed to the dendrites by using CF-LSM, GFP and VGluT2 immunoreactivities were further developed for electron microscopy by using immunogold/silver enhancement and immunoperoxidase/diaminobenzidine (DAB) methods, respectively. In contrast-inverted FIB-SEM images, silver precipitations and DAB deposits were observed as fine dark grains and diffuse dense profiles, respectively, indicating that these immunoreactivities were as easily recognizable as those in the transmission electron microscopy images. Furthermore, in the sites of interest, some appositions displayed synaptic specializations of an asymmetric type. Thus, the present method was useful in the three-dimensional analysis of immunocytochemically differentiated synaptic connections in the central neural circuit. (1648 chars). © 2013 Sonomura, Furuta, Nakatani, Yamamoto, Unzai, Matsuda, Iwai, Yamanaka, Uemura and Kaneko.