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Author:
Date:
2020-07-20
[Abstract] Members of the claudin family of tight junction proteins regulate paracellular permeability and modulate cell signaling. During junction remodeling, these proteins are selectively inserted into or retrieved from the tight junctions, but the control and coordination of these processes remain incompletely understood. Visualization of claudins allows the assessment of changes in their localization and abundance. We use the described protocol to stain claudin-2, but it can also be adapted to stain any tight junction protein. We found that using methanol for fixing allows the best preservation of claudin-2 both at the membrane and in cytoplasmic vesicles. Staining is done using a claudin-2 specific primary and a fluorescently labelled secondary antibody, along with DAPI to label nuclei. The ...
[摘要] [摘要 t] 紧密连接蛋白claudin家族的成员调节细胞旁通透性并调节细胞信号传导。在连接重塑过程中，这些蛋白被选择性地插入紧密连接或从紧密连接中检索出来，但是对这些过程的控制和协调仍不完全了解。claudins的可视化可以评估其定位和丰度的变化。我们使用所描述的方案对claudin-2染色，但它也可以用于染色任何紧密连接蛋白。我们发现使用甲醇进行固定可以使claudin-2在膜和细胞质囊泡中得到最佳保存。使用claudin-2特异性一抗和荧光标记的二抗以及与DAPI标记核一起进行染色。然后使用共聚焦显微镜对样品成像，并获得z堆栈，从而可以可视化连接和细胞内claudin-2。总的claudin-2信号可以在3D重建图像后使用Imaris 软件。
[背景 ] 紧密连接（TJ）是一种多蛋白复合物，位于连接上皮细胞的细胞间连接复合物的最顶端（Van Itallie和Anderson，2014）。这些结构产生通透性屏障和离子特异性旁细胞途径，维持apicobasal 极性，为各种重要功能提供输入并调节信号传导途径。位于TJs的蛋白可分为跨膜蛋白和相关的胞质蛋白（综述见（González-Mariscal ...
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