| In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin
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Author:
Date:
2020-08-05
[Abstract] In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da ...
[摘要] [摘要]为了使大脑正常工作，必须维持精心安排的体内平衡。为了帮助调节这种微妙的平衡，大脑已经形成了一种高度选择性的血脑屏障（BBB）。在正常情况下，血脑屏障将有害的血液传播物质排除在脑实质之外。然而，许多神经病理学条件可以破坏这一屏障，导致BBB通透性和随后的CNS功能障碍。了解BBB通透性的机制对于阐明各种神经系统疾病的病理学以及确定药物输送到中枢神经系统的方法至关重要。在这里，我们描述了几种在体内使用不同大小的示踪剂（包括外源性376da荧光素盐、66.5kDa牛血清白蛋白、70kDa右旋糖酐以及内源性160kDa小鼠IgG）来测量BBB在小鼠体内的通透性。当静脉注射时，这些物质会被BBB排除在健康大脑之外。然而，BBB功能障碍可使这些示踪剂进入大脑，这种积聚可通过分光光度法、荧光显微镜和免疫组织化学进行测量。我们还描述了一种利用产气荚膜梭菌epsilon毒素诱导BBB通透性的方法。最后，我们将简要讨论每种方法的优缺点及其适当的下游应用。
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| Immunofluorescent Staining of Claudin-2 in Cultured Kidney Tubular Cells
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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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| Co-immunostaining of ICAM-1, ICAM-2, and CD31 in Mouse Kidney Glomeruli
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Author:
Date:
2020-07-05
[Abstract] Glomerulonephritis (GN) is a common pathological condition in chronic kidney diseases that often leads to end stage renal failure. Mac-1 (CD11b/CD18)-mediated neutrophil, macrophage, and dendritic cell glomerular infiltration leading to cellular dysfunction and destruction is an important disease mechanism. The cellular distribution and dynamics of the expression of Mac-1 ligands ICAM-1 and ICAM-2 in GN have not been well studied because of the difficulties in tissue staining and colocalizing glomerular cells with surface antigens. To improve the visualization of cell surface marker and antigen expression in kidney compartments, we have devised an even but mild fixation procedure employing p-formaldehyde-lysine-periodate (PLP) perfusion. A large panel of antibodies (Ab) against cell ...
[摘要] [摘要 ] 肾小球肾炎（GN）是慢性肾脏疾病的常见病理状况，通常会导致晚期肾衰竭。Mac-1（CD11b / CD18）介导的嗜中性粒细胞，巨噬细胞和树突状细胞肾小球浸润导致细胞功能障碍和破坏是重要的疾病机制。由于在组织染色和肾小球细胞与表面抗原共定位方面存在困难，因此尚未对GN中Mac-1配体ICAM-1和ICAM-2的表达进行细胞分布和动力学研究。为了改善肾脏区室中细胞表面标志物和抗原表达的可视化，我们设计了一种均匀而温和的固定方法，即使用对甲醛-高赖氨酸-高碘酸盐（PLP）灌注。一大批针对细胞表面标志物的抗体（Ab）用于鉴定肾细胞类型和粘附分子。当使用共聚焦显微镜观察肾小球粘附分子染色时，发现内皮细胞特异性表达CD31，并且这些细胞组成性表达ICAM-2。尽管ICAM-1在稳态中不由肾小球内皮细胞表达，但在患有慢性GN和严重蛋白尿的小鼠中ICAM-1高度上调。VCAM-1是在白细胞迁移中重要的VLA-4配体，未在肾小球中表达。结果强调了ICAM-1在巨噬细胞和树突状细胞浸润中的重要性。cGN 。该报告将为产生高质量共聚焦图像以及鉴定和定量在不同肾区室和细胞类型中表达的受体和其他细胞抗原提供广泛适用的程序。
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