Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs).
Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs).
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DOI：
10.3389/fncel.2013.00054
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发表时间：
2013
影响因子：
5.3
通讯作者：
Derouiche A
中科院分区：
文献类型：
--
作者：
Haseleu J;Anlauf E;Blaess S;Endl E;Derouiche A
研究星形胶质细胞抗原的分布是特别困难的，当它们被定位在他们的罚款，外周星形胶质细胞过程（PAP），因为这些过程往往有一个直径相当的囊泡和小细胞器。最合适的技术是免疫电子显微镜，然而，这是一个耗时的过程。即使在高分辨率光学显微镜下，由于这些过程的小尺寸以及来自周围非神经胶质细胞中的抗原的覆盖，抗原定位也难以检测。然而，PAP经常显示与运动性和胶质-突触相互作用相关的抗原。在这里，我们描述了形态完整的神经胶质细胞（DIMIGs）的解离，允许明确的抗原定位使用落射荧光显微镜。通过应用木瓜蛋白酶处理和细胞离心分离器离心将星形胶质细胞从幼年（p13-15）小鼠皮层解离，以将细胞附着到载玻片上。因此，细胞及其包括PAP的完整过程以2D投影。整个过程需要2.5-3小时。我们通过形态计量学表明，DIMIGs（包括PAP）的直径与原位星形胶质细胞的直径相似。与细胞培养相反，从该程序获得的结果允许直接得出与以下相关的结论：（1）皮质星形胶质细胞中存在抗原，（2）亚细胞抗原分布，特别是当定位于PAP中时。详细的分辨率显示在组织的星形胶质细胞细胞骨架组件肌动蛋白，埃兹蛋白，微管蛋白和GFAP的示例性研究。连接蛋白43的分布与单个星形胶质细胞的过程树也进行了研究。
Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13–15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5–3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated.
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影响因子：
3.7
作者：
Haberlandt C;Derouiche A;Wyczynski A;Haseleu J;Pohle J;Karram K;Trotter J;Seifert G;Frotscher M;Steinhäuser C;Jabs R
通讯作者：
Jabs R
影响因子：
6.2
作者：
Ou, J. W.;Kumar, Y.;Alioua, A.;Sailer, C.;Stefani, E.;Toro, L.
通讯作者：
Toro, L.
影响因子：
4.8
作者：
Coco, S;Calegari, F;Verderio, C
通讯作者：
Verderio, C
影响因子：
6.2
作者：
Derouiche, A;Frotscher, M
通讯作者：
Frotscher, M
影响因子：
6.2
作者：
Anlauf, E;Derouiche, A
通讯作者：
Derouiche, A