Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.
Correlative analysis of immunoreactivity in confocal laser-scanning microscopy and scanning electron microscopy with focused ion beam milling.
复制标题
DOI：
10.3389/fncir.2013.00026
复制
发表时间：
2013
影响因子：
3.5
通讯作者：
Kaneko T
中科院分区：
文献类型：
--
作者：
Sonomura T;Furuta T;Nakatani I;Yamamoto Y;Unzai T;Matsuda W;Iwai H;Yamanaka A;Uemura M;Kaneko T
近年来，通过使用扫描电子显微镜（SEM）结合聚焦离子束（FIB）铣削（FIB-SEM），以最小的努力实现了脑超微结构的三维重建。免疫组织化学染色在电子显微镜（EM）中的应用提供了很大的优势，因为感兴趣的分子特异性地定位在超微结构中。因此，我们应用免疫细胞化学的FIB-扫描电镜和相关的免疫反应，在共聚焦激光扫描显微镜（CF-LSM）。使用重组病毒载体可视化大鼠新纹状体中中等大小的多刺神经元的树突，该载体以高尔基体染色样方式用膜靶向GFP标记感染的神经元。此外，丘脑纹状体传入终末免疫标记与Cy 5荧光囊泡谷氨酸转运蛋白2（VGluT 2）。在用CF-LSM检测树突末端的位置后，分别用免疫金/银增强法和免疫过氧化物酶/二氨基联苯胺（DAB）法进一步检测EM的GFP和VGluT 2免疫反应性。在对比度倒置的FIB-SEM图像中，观察到银沉淀和DAB沉积物分别为细暗颗粒和弥散致密轮廓，表明这些免疫反应性与透射电子显微镜（TEM）图像中的免疫反应性一样容易识别。此外，在感兴趣的网站，一些并置显示突触专业化的不对称类型。因此，本发明的方法是有用的，在三维分析的免疫细胞化学分化的突触连接在中枢神经回路。
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.
登录
查看更多内容
影响因子：
2.5
作者：
Darcy, Michael J.;Calvin, Kate;Ouimet, Charles C.
通讯作者：
Ouimet, Charles C.
影响因子：
2.9
作者：
Merchan-Perez, Angel;Rodriguez, Jose-Rodrigo;DeFelipe, Javier
通讯作者：
DeFelipe, Javier
影响因子：
3.3
作者：
Hioki, H;Fujiyama, F;Kaneko, T
通讯作者：
Kaneko, T
影响因子：
16.2
作者：
Sugita, S;Han, WP;Südhof, TC
通讯作者：
Südhof, TC
影响因子：
3.2
作者：
GRAHAM, RC;KARNOVSKY, MJ
通讯作者：
KARNOVSKY, MJ