Puente Nagore, Río Itziar Bonilla-Del, Achicallende Svein, Nahirney Patrick C, Grandes Pedro
Department of Neurosciences, Faculty of Medicine and Nursing, University of the Basque Country UPV/EHU, E-48940 Leioa, Spain.
Achucarro Basque Center for Neuroscience, Science Park of the UPV/EHU, Leioa, Spain.
Bio Protoc. 2019 Jan 20;9(2):e3145. doi: 10.21769/BioProtoc.3145.
Activation of type 1 cannabinoid (CB) receptors by endogenous, exogenous (cannabis derivatives) or synthetic cannabinoids (, CP 55.940, Win-2) has a wide variety of behavioral effects due to the presence of CB receptors in the brain. hybridization and immunohistochemical techniques have been crucial for defining the CB receptor expression and localization at the cellular level. Nevertheless, more advanced methods are needed to reveal the precise topography of CB receptors in the brain, especially in unsuspected sites such as other cell types and organelles with low receptor expression (, glutamatergic neurons, astrocytes, mitochondria). High-resolution immunoelectron microscopy provides a more precise detection method for the subcellular localization of CB receptors in the brain. Herein, we describe a single pre-embedding immunogold method for electron microscopy based on the use of specific CB receptor antibodies and silver-intensified 1.4 nm gold-labeled Fab' fragments, and a combined pre-embedding immunogold and immunoperoxidase method that employs biotinylated secondary antibodies and avidin-biotin-peroxidase complex for the simultaneous localization of CB receptors and protein markers of specific brain cells or synapses (, GFAP, GLAST, IBA-1, PSD-95, gephyrin). In addition, a post-embedding immunogold method is also described and compared to the pre-embedding labeling procedure. These methods provide a relatively easy and useful approach for revealing the subcellular localization of low amounts of CB receptors in glutamatergic synapses, astrocytes, neuronal and astrocytic mitochondria in the brain.
由于大脑中存在1型大麻素(CB)受体,内源性、外源性(大麻衍生物)或合成大麻素(如CP 55.940、Win-2)对其激活会产生多种行为效应。杂交和免疫组织化学技术对于在细胞水平上确定CB受体的表达和定位至关重要。然而,需要更先进的方法来揭示大脑中CB受体的精确拓扑结构,特别是在未被怀疑的部位,如受体表达较低的其他细胞类型和细胞器(如谷氨酸能神经元、星形胶质细胞、线粒体)。高分辨率免疫电子显微镜为大脑中CB受体的亚细胞定位提供了一种更精确的检测方法。在此,我们描述了一种基于使用特异性CB受体抗体和银增强的1.4 nm金标记Fab'片段的用于电子显微镜的单预包埋免疫金方法,以及一种联合预包埋免疫金和免疫过氧化物酶方法,该方法采用生物素化二抗和抗生物素蛋白-生物素-过氧化物酶复合物来同时定位CB受体和特定脑细胞或突触的蛋白质标记物(如GFAP、GLAST、IBA-1、PSD-95、gephyrin)。此外,还描述了一种包埋后免疫金方法并将其与预包埋标记程序进行比较。这些方法为揭示大脑中谷氨酸能突触、星形胶质细胞、神经元和星形胶质细胞线粒体中少量CB受体的亚细胞定位提供了一种相对简单且有用的方法。
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