title: Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin.
abstract: Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.
authors: Psychoneuroimmunology Laboratory, Department of Psychosomatic Medicine and Psychotherapy (FRR, BR, ST, EMJP)
authors: Frank R
authors: FR
authors: Rommel
authors: Psychoneuroimmunology Laboratory, Department of Psychosomatic Medicine and Psychotherapy (FRR, BR, ST, EMJP)
authors: Badrinarayanan
authors: B
authors: Raghavan
authors: Institute of Anatomy and Cell Biology, Justus Liebig University, Giessen, Germany (RP, WK)
authors: Renate
authors: R
authors: Paddenberg
authors: Institute of Anatomy and Cell Biology, Justus Liebig University, Giessen, Germany (RP, WK)
authors: Wolfgang
authors: W
authors: Kummer
authors: Psychoneuroimmunology Laboratory, Department of Psychosomatic Medicine and Psychotherapy (FRR, BR, ST, EMJP)
authors: Susanne
authors: S
authors: Tumala
authors: Institute of Biochemistry, Justus Liebig University, Giessen, Germany (GL)
authors: Günter
authors: G
authors: Lochnit
authors: Department of Dermatology, University Hospital Giessen, Giessen, Germany (UG)
authors: Uwe
authors: U
authors: Gieler
authors: Psychoneuroimmunology Laboratory, Department of Psychosomatic Medicine and Psychotherapy (FRR, BR, ST, EMJP)|Charité Center 12 for Internal Medicine and Dermatology, Universitätsmedizin Charité, Berlin, Germany (EMJP)
authors: Eva M J
authors: EM
authors: Peters
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D015151:Immunoblotting; D007150:Immunohistochemistry; D008407:Mast Cells / Q000378:metabolism; D018345:Mice, Knockout; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012867:Skin / Q000378:metabolism*; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000199:Actins; D000906:Antibodies; D012333:RNA, Messenger; D043587:Receptor, Muscarinic M3; D064569:alpha7 Nicotinic Acetylcholine Receptor; D010751:Phosphopyruvate Hydratase
keywords: atopic dermatitis-like allergic dermatitis (AlD); muscarinic acetylcholine receptor 3 (Chrm3); nicotinic acetylcholine receptor α7 (Chrna7); non-neuronal cholinergic system (NNCS); stress