Biochemical and histochemical evidence of nonspecific binding of α7nAChR antibodies to mouse brain tissue
Biochemical and histochemical evidence of nonspecific binding of α7nAChR antibodies to mouse brain tissue
复制标题
DOI：
10.1369/jhc.4a6319.2004
复制
发表时间：
2004-10-01
影响因子：
3.2
通讯作者：
Gordon, MN
中科院分区：
文献类型：
--
作者：
Herber, DL;Severance, EG;Gordon, MN
α - 7烟碱乙酰胆碱受体参与学习和记忆，并与阿尔茨海默病和精神分裂症的病理有关。检测alpha7亚基可以通过免疫检测或a-bungarotoxin结合技术来完成。使用几种市售抗体，遵循免疫组织化学和免疫印迹的标准方案。评估了各种小鼠，包括非转基因、APP、PS1、APP+PS1和alpha7敲除。淀粉样蛋白沉积小鼠的初步结果显示，除了预期的神经元染色外，还有α 7免疫标记的星形胶质细胞。随后对alpha7基因敲除小鼠海马内注射脂多糖（LPS）的研究表明，alpha7抗体对神经元和星形胶质细胞的标记都是非特异性的。在小鼠脑蛋白的Western blots上，在alpha7敲除小鼠中，针对alpha7亚基的抗体检测到的条带都没有减少。虽然发现了与lps相关的一些条带的表达变化，但这些也不受小鼠的alpha7基因型的影响。一般来说，这些抗体的Western染色模式显示很少重叠带。这些免疫检测数据与基因分型结果和mRNA分析形成对比，后者证实了基因敲除中alpha7等位基因的破坏和alpha7信息的缺乏。这些发现提示，当使用几种市售的alpha7烟碱受体抗体时，解释结果时要谨慎。
Alpha 7 nicotinic acetylcholine receptors are involved in learning and memory, and are implicated in the pathology of Alzheimer's disease and schizophrenia. Detection of alpha7 subunits can be accomplished via immunodetection or a-bungarotoxin-binding techniques. Standard protocols for immunohistochemistry and Western blotting were followed using several commercially available antibodies. Various mice were evaluated, including non-transgenics, APP, PS1, APP+PS1, and alpha7 knockouts. Initial results with amyloid-depositing mice revealed alpha7 immunolabeled astrocytes, in addition to expected neuronal staining. Subsequent studies with intrahippocampal injections of lipopolysaccharicle (LPS) into alpha7 knockout mice showed that both neuronal and astrocytic labeling by alpha7 antibodies was nonspecific. On Western blots of mouse brain proteins, none of the bands detected with antibodies directed against alpha7 subunits diminished in the alpha7 knockout mice. Although LPS-related changes in the expression of some bands were found, these also were unaffected by the alpha7 genotype of the mice. In general, the Western staining patterns for these antibodies revealed few overlapping bands. These immunodetection data are in contrast to genotyping results and mRNA analyses that confirmed the disruption of the alpha7 allele and lack of alpha7 message in the knockouts. These findings suggest caution in interpreting results when using several commercially available alpha7 nicotinic receptor antibodies.