Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.
Combining patch-clamping of cells in brain slices with immunocytochemical labeling to define cell type and developmental stage.
复制标题
将脑切片中细胞的膜片钳与免疫细胞化学标记相结合，以确定细胞类型和发育阶段。
DOI：
10.1038/nprot.2006.261
复制
发表时间：
2006
期刊：
影响因子：
14.8
通讯作者：
Attwell, David
中科院分区：
文献类型：
--
作者：
Karadottir, Ragnhildur;Attwell, David
在神经科学中，将膜片钳与同一细胞中的蛋白质鉴定相结合变得越来越重要，以确定神经元或神经胶质细胞的哪个亚型或发育阶段被记录，并将测量的膜电流归因于表达的离子通道或受体。在这里，我们描述了一个协议，以实现这一目标时，研究细胞在急性脑切片，抗体渗透到差，并洗涤剂透化不能使用时，使用抗体，识别脂质成分，如O 4硫苷脂。该方法避免了对电生理记录切片的切除的需要。它采用在全细胞记录期间用荧光染料填充细胞，以允许随后的细胞定位，然后用最多3种抗体固定和自由浮动切片标记，这些抗体可以识别膜、核或胞质蛋白。通过实践，大约80%的膜片钳细胞可以被回收并以这种方式鉴定其蛋白质。整个方案可在3-4天内完成。
In neuroscience, combining patch-clamping with protein identification in the same cell is becoming increasingly important to define which subtype or developmental stage of a neuron or glial cell is being recorded from, and to attribute measured membrane currents to expressed ion channels or receptors. Here we describe a protocol to achieve this when studying cells in acute brain slices, which antibodies penetrate poorly into, and for which detergent permeabilization cannot be used when using antibodies that recognize lipid components such as O4 sulfatide. The method avoids the need for resectioning of the electrophysiologically recorded slices. It employs filling of the cell with a fluorescent dye during whole-cell recording, to allow subsequent localization of the cell, followed by fixation and free floating section labelling with up to 3 antibodies, which may recognize membrane, nuclear or cytosolic proteins. With practice, ∼80% of patch-clamped cells can be retrieved and have their proteins identified in this way. The entire protocol can be completed in 3-4 days.
登录
查看更多内容
影响因子：
64.8
作者：
Bergles, DE;Roberts, JDB;Jahr, CE
通讯作者：
Jahr, CE
影响因子：
3.4
作者：
Ullensvang, K;Lehre, KP;Danbolt, NC
通讯作者：
Danbolt, NC
影响因子：
5.3
作者：
Mallon, BS;Shick, HE;Macklin, WB
通讯作者：
Macklin, WB
影响因子：
2.8
作者：
Grateron, L;Cebada-Sanchez, S;Insausti, R
通讯作者：
Insausti, R
影响因子：
56.9
作者：
Gupta, A;Wang, Y;Markram, H
通讯作者：
Markram, H