A method for triple fluorescence labeling with Vicia villosa agglutinin, an anti-parvalbumin antibody and an anti-G-protein-coupled receptor antibody

Abstract The aim of the original study [S.B. Bausch, C. Chavkin, Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum, Dev. Brain Res., 97, 1996, 169–177] [3] was to develop a method for identifying a subset of mu opioid receptor-expressing interneurons in the rat subiculum for electrophysiological studies. Previous studies had shown that a subset of parvalbumin-positive neurons in the rat subiculum could be labeled with the lectin, Vicia villosa agglutinin (VVA) [C.T. Drake, K.A. Mulligan, T.L. Wimpey, A. Hendrickson, C. Chavkin, Characterization of Vicia villosa agglutinin-labeled GABAergic neurons in the hippocampal formation and in acutely dissociated hippocampus, Brain Res., 554, 1991, 176–185] [11] , and that mu opioid receptor immunoreactivity (-IR) and parvalbumin-IR were colocalized in a subset of neurons in the hippocampus and dentate gyrus [S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin and a G-protein-coupled inwardly rectifying potassium channel in the rodent brain, Analgesia, 1, 1995, 282–285] [2] . We hypothesized that a subset of mu opioid receptor-expressing neurons in the subiculum also would express the calcium binding protein, parvalbumin, and could be labeled with VVA. Labeling of live neurons with VVA [11] then could be used to identify these neurons. This protocol was designed to triple-label neurons expressing the mu opioid receptor, parvalbumin and the carbohydrate group, N -acetylgalactosamine (which binds VVA [S.E. Tollefsen, R. Kornfeld, The B4 lectin from Vicia villosa seeds interacts with N -acetylgalactosamine residues α -linked to serine or threonine residues in cell surface glycoproteins, J. Biol. Chem., 258, 1983, 5172–5176][M.P. Woodward, W.W. Young, R.A. Bloodgood, Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation, J. Immunol. Methods, 78, 1985, 143–153] [25, 29] ). VVA labeling and immunocytochemistry with an affinity-purified anti-mu opioid receptor antibody [S.B. Bausch, T.A. Patterson, M.U. Ehrengruber, H.A. Lester, N. Davidson, C. Chavkin, Colocalization of mu opioid receptors with GIRK1 potassium channels in rat brain: an immunocytochemical study, Recept. Channels, 3, 1995, 221–241] [4] and an anti-parvalbumin antibody [M.R. Celio, W. Baier, L. Scharer, P.A. de Viragh, C. Gerday, Monoclonal antibodies directed against the calcium binding protein parvalbumin, Cell Calcium, 9, 1988, 81–86] [8] were used to accomplish this goal. Immunofluorescence was used as the detection method; visualization was accomplished with three fluorophores with different excitation/emission spectra and a one laser confocal microscope. This protocol can be modified easily to triple-label neurons for other carbohydrate groups and proteins. Themes: Cell biology Topics: Staining, tracing, and imaging techniques