Validation of a selective serotonin 5-HT2C receptor antibody for utilization in fluorescence immunohistochemistry studies
Validation of a selective serotonin 5-HT2C receptor antibody for utilization in fluorescence immunohistochemistry studies
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DOI：
10.1016/j.brainres.2005.09.050
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发表时间：
2005-11-30
期刊：
影响因子：
2.9
通讯作者：
Cunningham, KA
中科院分区：
文献类型：
--
作者：
Bubar, MJ;Seitz, PK;Cunningham, KA
尽管放射性配体结合研究表明5-羟色胺5-HT 2C受体（5-HT 2CR）在整个大脑中广泛表达，但更详细地了解5-HT 2CR在不同神经元群体中的分布将有助于理解该受体的作用机制。双标记免疫组织化学程序可用于检查特定神经元群体内受体的定位。然而，为了进行这样的研究，首先需要检查两种市售抗5-HT 2CR抗体[来自圣克鲁斯（SC）和BD PharMingen（PH）]的效用和特异性。在雄性Sprague-Dawley大鼠中，这两种抗体在选择用于研究的整个脑区（腹侧被盖区，这是多巴胺mesocorticobens“奖励”途径的起源）产生广泛的免疫反应性（IR）。用SC和PH 5-HT 2CR抗体的共标记证明两种抗体的IR大部分重叠。然而，SC 5-HT 2CR IR在IR细胞体内更集中，并且在测定中比PH 5-HT 2CR IR更一致。因此，选择SC 5-HT 2CR抗体用于后续研究。当.在5-HT 2CR敲除小鼠与野生型小鼠中检查，SC 5-HT 2CR抗体在野生型而不是5-HT 2CR敲除小鼠中产生广泛的IR。此外，5-HT 2CR-IR不存在于已知缺乏5-HT 2AR或5-HT 2CR的天然CHO细胞或用5-HT 2AR转染的CHO细胞中。因此，这些研究表明，SC 5-HT 2CR抗体在啮齿动物脑中产生对5-HT 2CR相对于5-HT 2AR的可靠染色选择性，因此适用于未来的免疫荧光5-HT 2CR定位研究。(C)2005 Elsevier B. V.保留所有权利。
Although radioligand binding studies have shown that the serotonin 5-HT2C receptor (5-HT2CR) is widely expressed throughout the brain, more detailed knowledge of 5-HT2CR distribution within different neuronal populations will aid in understanding the mechanisms through which this receptor acts. Double-label immunohistochemical procedures can be utilized to examine the localization of receptors within specific neuronal populations. In order to conduct such studies, however, it was first necessary to examine the utility and specificity of two commercially available anti-5-HT2CR antibodies [from Santa Cruz (SC) and BD PharMingen (PH)]. In male Sprague-Dawley rats, both antibodies produced widespread immunoreactivity (IR) throughout the brain area chosen for study, the ventral tegmental area, which is the origin of the dopamine mesocorticoaccumbens "reward" pathway. Co-labeling with the SC and PH 5-HT2CR antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT2CR IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT2CR IR. Thus, the SC 5-HT2CR antibody was chosen for subsequent studies. When. examined in 5-HT2CR knockout vs. wild-type mice, the SC 5-HT2CR antibody produced widespread IR in wild-type, but not 5-HT2CR knockout, mice. In addition, 5-HT2CR-IR was not present in either native CHO cells, known to be devoid of 5-HT2AR or 5-HT2CR, or in CHO cells transfected with the 5-HT2AR. Thus, these studies suggest that the SC 5-HT2CR antibody produces reliable staining selective for 5-HT2CR vs. 5-HT2AR in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT2CR localization studies. (C) 2005 Elsevier B.V. All rights reserved.