Metabolic activity in antigenically identified neurons: a double labeling method for high-resolution 2-deoxyglucose and immunohistochemistry.
Metabolic activity in antigenically identified neurons: a double labeling method for high-resolution 2-deoxyglucose and immunohistochemistry.
复制标题
抗原识别神经元的代谢活性：高分辨率 2-脱氧葡萄糖和免疫组织化学的双标记方法。
DOI：
10.1016/0165-0270(96)00063-5
复制
发表时间：
1996
影响因子：
3
通讯作者：
McCasland,JS
中科院分区：
文献类型：
--
作者：
McCasland,JS
Sokoloff等人的2-脱氧葡萄糖（2DG）组织化学方法（（1977）J. Neurochem.，28：897-916）已被广泛用于绘制大脑代谢活动，但缺乏足够的分辨率来查看单个神经元。原始方法的修改已经实现了细胞分辨率与乳剂放射自显影和组织直接贴附到放射自显影，但区分清楚的抗原标记物确定的神经元群体之间的能力一直缺乏。提出了一种方法，这使得它有可能，第一次，研究的每一个化学标记的神经元在感兴趣的神经系统中的代谢活动。该程序结合了高分辨率2-脱氧葡萄糖技术和谷氨酸脱羧酶（GAD）或其他抗原的免疫染色。该方法通过向切片后与组织接触的所有溶液中添加糖原，在免疫组织化学处理期间稳定标记。添加糖原的假定作用是限制含有2-脱氧葡萄糖标记的未固定大分子的扩散或净流出。当与GAD抗体一起使用来标记推定的GABA能抑制性中间神经元时，该程序可以直接评估大脑皮质回路中的抑制程度。这种双标记程序的详细协议，沿着与啮齿动物体感皮层的胡须相关的桶字段中获得的标记模式的例子。
The 2-deoxyglucose (2DG) histochemical method of Sokoloff ea al. ((1977) J. Neurochem., 28: 897–916) has been widely used to map metabolic activity in the brain, but lacks sufficient resolution to view individual neurons. Modifications of the original method have achieved cellular resolution with emulsion autoradiography and tissue apposed directly to the autoradiogram, but the ability to discriminate clearly between populations of neurons identified by antigenic markers has been lacking. A method is presented which makes it possible, for the first time, to study the metabolic activity of every immunohistochemically labeled neuron in a neural system of interest. The procedure combines a high-resolution 2-deoxyglucose technique and immunostaining for glutamate decarboxylase (GAD) or other antigens. The method stabilizes label during immunohistochemical processing by the addition of glycogen to all solutions that come into contact with tissue after sectioning. The postulated effect of the added glycogen is to limit diffusion or net efflux of unfixed macromolecules which contain 2-deoxyglucose label. When used with a GAD antibody to label putative GABAergic inhibitory interneurons, the procedure allows a direct assessment of the extent of inhibition in the circuitry of cerebral cortex. A detailed protocol for this double-labeling procedure is presented, along with examples of the labeling patterns obtained in the whisker-related barrel field of rodent somatosensory cortex.