Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice

摘要

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). Here, we assessed the specificity of 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 8 of them in 21 different protocols, resulting in a total number of 456 antibody/protocol combinations. Each of them was tested at 4 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 unlabeled in the preabsorption 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 preabsorption control in wild-type mice suggested specificity. Hence, the present data demonstrate the unpleasant fact that reliable immunohistochemical localization of MR subtypes with antibodies is the exception rather than the rule. Immunohistochemical detection of MR subtype localization in tissue sections of peripheral organs is limited to the M2R subtype utilizing the most commonly used methodological approaches.