Jositsch Gitte, Papadakis Tamara, Haberberger Rainer V, Wolff Miriam, Wess Jürgen, Kummer Wolfgang
Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Aulweg 123, 35385, Giessen, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2009 Apr;379(4):389-95. doi: 10.1007/s00210-008-0365-9. Epub 2008 Oct 31.
Acetylcholine (ACh) is a major regulator of visceral function exerting pharmacologically relevant effects upon smooth muscle tone and epithelial function via five types of muscarinic receptors (M1R-M5R). In this paper, we assessed the specificity of muscarinic receptor (MR) antibodies in immunohistochemical labelling on tissue sections by analysing specimens from wild-type and respective gene-deficient mice. Of 24 antibodies evaluated in this study, 16 were tested at 18 different conditions each, and eight of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at four antibody dilutions at minimum, so that finally, at least 1,824 conditions were evaluated. For each of them, dorsal root ganglia, urinary bladder and cross-sections through all thoracic viscera were investigated. In all cases where the antigen was available, at least one incubation condition was identified in which only select cell types were immunolabelled in the positive control but remained unlabelled in the pre-absorption control. With two exceptions (M2R antibodies), however, all antibodies produced identical immunohistochemical labelling patterns in tissues taken from corresponding gene-deficient mice even when the pre-absorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localisation of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localisation in tissue sections of peripheral organs is limited to the M2R subtype utilising the most commonly used methodological approaches.
乙酰胆碱（ACh）是内脏功能的主要调节因子，通过五种毒蕈碱受体（M1R - M5R）对平滑肌张力和上皮功能发挥药理学相关作用。在本文中，我们通过分析野生型和相应基因缺陷小鼠的标本，评估了毒蕈碱受体（MR）抗体在组织切片免疫组织化学标记中的特异性。在本研究评估的24种抗体中，16种在18种不同条件下进行了测试，其中8种在21种不同方案下进行了测试，总共产生了456种抗体/方案组合。每种组合至少在四种抗体稀释度下进行测试，因此最终至少评估了1824种条件。对于每种组合，研究了背根神经节、膀胱以及所有胸段内脏的横截面。在所有抗原存在的情况下，至少确定了一种孵育条件，其中在阳性对照中只有特定细胞类型被免疫标记，而在预吸收对照中保持未标记。然而，除了两个例外（M2R抗体），即使野生型小鼠的预吸收对照显示出特异性，所有抗体在相应基因缺陷小鼠的组织中产生的免疫组织化学标记模式相同。因此，目前的数据表明了一个令人不快的事实，即使用抗体对MR亚型进行可靠的免疫组织化学定位是例外而非常规。利用最常用的方法，在外周器官组织切片中对MR亚型定位的免疫组织化学检测仅限于M2R亚型。
