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

Abstract

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.

How reliable are G-protein-coupled receptor antibodies?

“…the N-or C-terminal tails may have a better chance for selectivity. However, some findings in this issue of the Journal demonstrate that N-and C-terminal-directed antibodies similarly lack specificity as those directed against more conserved receptor domains (Jensen et al 2009;Jositsch et al 2009). Irrespective of such considerations, these problems suggest that many previously published data, including some in this Journal (Otsuka et al 2008;van Diepen et al 2008), may require re-interpretation.…”

“…However, the problem apparently goes well beyond the homology of closely related GPCR subtypes. For example, airways express only the M 2 and M 3 subtype of muscarinic receptors (Michel and Parra 2008) but double-knock-out mice lacking both subtypes still stained positive for M 2 and M 3 receptor antibodies (Jositsch et al 2009). Similarly, triple knock-out mice lacking all three α 1 -adrenoceptor subtypes show similar staining patters as wild-type mice (Jensen et al 2009).…”

“…A second soft criterion used in the past is that antibodies against different subtypes of a given receptor exhibit distinct staining patters in tissues. However, studies with both knock-out mice (Everaerts et al 2009;Jensen et al 2009;Jositsch et al 2009;Lu and Bartfai 2009;Pradidarcheep et al 2008) or immunoblots comparing staining to cloned receptor subtypes (Bodei et al 2009;Hamdani and van der Velden 2009;Pradidarcheep et al 2009) demonstrate that this also is insufficient to prove selectivity. Rather we propose that at least one of the following four conditions must be met to consider an antiserum to indeed be selective.…”

“…Several studies demonstrate that many available antibodies fail to meet this test (Everaerts et al 2009;Jensen et al 2009;Jositsch et al 2009;Lu and Bartfai 2009;Pradidarcheep et al 2008). Secondly, animals or cell lines can be treated with genetic tools to knock-down expression of a given receptor, e.g.…”

“…In this issue of the Journal, seven groups of investigators report a lack of selectivity of a large number of receptor antibodies for the claimed target receptor. This includes antibodies against subtypes of α 1 -adrenoceptors (Jensen et al 2009), β-adrenoceptors (Hamdani and van der Velden 2009;Pradidarcheep et al 2009), dopamine receptors (Bodei et al 2009), muscarinic receptors (Jositsch et al 2009;Pradidarcheep et al 2009), and galanin receptors (Lu and Bartfai 2009). An additional report shows similar problems with antibodies against receptors from the family of ligand-gated ion channels, specifically the TRPV1 receptors (Everaerts et al 2009), although other antibodies directed against these receptors apparently are selective by hard criteria (Charrua et al 2009).…”