Precision for Medicine, Redwood City, CA, USA.
Oxford Biotherapeutics Inc., San Jose, CA, USA.
Methods Mol Biol. 2024;2711:257-262. doi: 10.1007/978-1-0716-3429-5_21.
The blood-brain barrier (BBB) is a highly complex and dynamic microvascular barrier that protects the brain parenchyma from the entry of pathogens, toxins, and other macromolecules and is a critical structure that helps to maintain brain homeostasis. The BBB is formed mainly by brain capillary endothelial cells and perivascular astrocytes and pericytes. One of the primary properties of the BBB is a tight regulation of paracellular permeability due to the presence of tight junctional complexes (also, adherens and gap junctions) between the neighboring microvascular endothelial cells. Alterations in the assembly of the tight junctions impair BBB properties, particularly influenced barrier integrity and permeability. The tight junctions of the BBB are mainly composed of proteins including claudins, occludin, and zonula occludens-1 (ZO-1). Zonula occludens-1 binds to the actin cytoskeleton, and its localization provides valuable information on the status of BBB integrity and permeability. Immunofluorescence localization of ZO-1 and/or other tight junction proteins is a reliable indicator of barrier integrity and permeability in microvascular endothelial cells. In microvascular endothelial cells, f-actin stress fiber formation significantly influences the rate and size of the inter-endothelial cell gap that form as cells retract from their borders. Rhodamine phalloidin is a popular conjugate used as a fluorescent label for f-actin. Herein, we describe the procedures for ZO-1 immunofluorescence and f-actin labeling followed by confocal microscopic imaging to determine barrier integrity and tight junction organization in brain microvascular endothelial cells in vitro.
血脑屏障（BBB）是一种高度复杂和动态的微血管屏障，可保护脑实质免受病原体、毒素和其他大分子的进入，是帮助维持脑内环境稳定的关键结构。BBB 主要由脑毛细血管内皮细胞和周细胞组成。BBB 的主要特性之一是通过相邻微血管内皮细胞之间存在紧密连接（黏附连接和缝隙连接）来严格调节细胞旁通透性。紧密连接组装的改变会损害 BBB 的特性，特别是影响屏障的完整性和通透性。BBB 的紧密连接主要由包括闭合蛋白、occludin 和 zonula occludens-1（ZO-1）在内的多种蛋白质组成。ZO-1 与肌动蛋白细胞骨架结合，其定位为 BBB 完整性和通透性的状态提供了有价值的信息。ZO-1 和/或其他紧密连接蛋白的免疫荧光定位是微血管内皮细胞中屏障完整性和通透性的可靠指标。在微血管内皮细胞中，f-肌动蛋白应力纤维的形成显著影响细胞从边界缩回时形成的内皮细胞间隙的大小和速度。罗丹明鬼笔环肽是一种常用的荧光标记物，用于 f-肌动蛋白。本文描述了 ZO-1 免疫荧光和 f-肌动蛋白标记的程序，随后进行共聚焦显微镜成像，以确定体外脑微血管内皮细胞的屏障完整性和紧密连接组织。
