Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures
Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures
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DOI：
10.1111/j.1471-4159.2007.04498.x
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发表时间：
2007-07-01
影响因子：
4.7
通讯作者：
Wevers, A.
中科院分区：
文献类型：
--
作者：
Moser, N.;Mechawar, N.;Wevers, A.
烟碱型乙酰胆碱受体在许多认知过程中起着重要作用，也在一些衰弱的中枢神经系统(CNS)疾病中起着重要作用。为了充分阐明烟碱型乙酰胆碱受体在中枢神经系统功能和功能障碍中的不同作用，对其细胞和亚细胞定位的详细了解是必不可少的。到目前为止，准确定位中枢神经系统烟碱型乙酰胆碱受体的方法主要依赖于抗受体亚单位抗体的使用。虽然免疫组织学和免疫印迹法获得的数据与配基结合研究基本一致，但仍然存在一些差异，特别是与电生理学发现的差异。在这种情况下，烟碱型乙酰胆碱受体亚单位缺陷小鼠应该是测试亚单位导向抗体特异性的理想工具。在这里，我们使用标准的免疫组织化学和免疫印迹方法来检测针对α3-、α4-、α7-、β2-和β4-烟碱型乙酰胆碱受体亚单位的抗体在各自的基因敲除小鼠的脑组织中产生。出乎意料的是，对于每一种被测试的抗体，野生型和基因敲除小鼠的免疫反应性是相同的。这些数据表明，在常用的条件下，这些抗体不适合免疫定位。因此，对于用来可视化大脑中的烟碱型乙酰胆碱受体的实验方法，应该特别谨慎。
Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti-receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit-deficient mice should be ideal tools for testing the specificity of subunit-directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the alpha 3-, alpha 4-, alpha 7-, beta 2-, and beta 4-nicotinic acetylcholine receptor subunits on brain tissues of the respective knock-out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild-type and knock-out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.