Immunohistochemical Methods for the Study of the Expression of Low-Affinity Monoamine Transporters in the Brain
Immunohistochemical and molecular methods, such as indirect immunofluorescence and in situ hybridization, have proven irreplaceable in providing an accurate vision of the precise localization of transporters as they occur within their native anatomical context in mammalian central nervous system, an essential prerequisite for understanding their function in vivo. Low-affinity monoamine transporters like organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT) represent important yet poorly characterized components of aminergic neurotransmission in the brain. Contrarily to the classical high-affinity reuptake transporters, these low-affinity monoamine transporters are not restricted to the presynaptic terminals, but distributed diffusely in cells of numerous brain areas, in particular of aminergic projection regions. In this chapter, we will provide a detailed description of well-established procedures for diaminobenzidine (DAB) immunohistochemistry and immunofluorescence successfully applied to rodent brain. We also detail immunohistochemistry in situ hybridization co-labeling, a valuable approach which couples the sensitive detection of the cells expressing transporter mRNAs in brain sections with the immunohistochemical identification of these cells. We discuss conditions that can improve transporter detection and identify critical methodological steps in these protocols.
Key wordsImmunohistochemistry Immunofluorescence Low-affinity monoamine transporters Central nervous system
We gratefully acknowledge the contribution of several former team members for the development and application of the methods described, in particular Vincent Vialou, Alexandre Bacq, Laure Balasse, and Quentin Perrenoud. Financial support was provided by the Institut National de la Santé et la Recherche Médicale (Inserm) and Fondation de France and fellowships from the French Ministry for Research, the Fondation pour la Recherche Médicale, and the Société Française de Pharmacologie et Thérapeutique.