Villaseñor Roberto, Collin Ludovic
Roche Pharma Research and Early Development (pRED), Neuroimmunology, Roche Innovation Center Basel.
Roche Pharma Research and Early Development (pRED), Neuroimmunology, Roche Innovation Center Basel;
J Vis Exp. 2017 Nov 16(129):56407. doi: 10.3791/56407.
The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.
血脑屏障（BBB）是一个动态的多细胞界面，可调节循环系统与大脑之间分子的运输。跨血脑屏障的转胞吞作用调节激素、代谢物和治疗性抗体向脑实质的递送。在这里，我们展示了一种方案，该方案将游离切片的免疫荧光与激光扫描共聚焦显微镜和图像分析相结合，以可视化血脑屏障处内皮细胞内的亚细胞器。将该数据集与3D图像分析软件相结合，可以对毛细血管体积和表面积以及血脑屏障处细胞内细胞器的数量和强度进行半自动分割和定量。检测细胞内囊泡中的小鼠内源性免疫球蛋白（IgG）并在血脑屏障处对其进行定量，以说明该方法。该方案有可能应用于研究体内控制不同分子血脑屏障转胞吞作用的机制。
