title: 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 the brain has been realized with minimal effort by using scanning electron microscopy (SEM) combined with focused ion beam (FIB) milling (FIB-SEM). Application of immunohistochemical staining in electron microscopy (EM) 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 EM 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 (TEM) 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.
authors: Department of Anatomy for Oral Sciences, Graduate School of Medical and Dental Sciences, Kagoshima University Kagoshima, Japan.
authors: Takahiro
authors: T
authors: Sonomura
authors: Takahiro
authors: T
authors: Furuta
authors: Ikuko
authors: I
authors: Nakatani
authors: Yo
authors: Y
authors: Yamamoto
authors: Tomo
authors: T
authors: Unzai
authors: Wakoto
authors: W
authors: Matsuda
authors: Haruki
authors: H
authors: Iwai
authors: Atsushi
authors: A
authors: Yamanaka
authors: Masanori
authors: M
authors: Uemura
authors: Takeshi
authors: T
authors: Kaneko
mesh_terms: D000818:Animals; D007473:Ion Channels / Q000502:physiology; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D017072:Neostriatum / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure*; D009415:Nerve Net / Q000737:chemistry / Q000166:cytology / Q000648:ultrastructure*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D007473:Ion Channels
keywords: confocal laser-scanning microscope; focused ion beam; immunocytochemistry; neostriatum; neural circuit; scanning electron microscopy; synapse; three-dimensional reconstruction