Assessing CB1 Expression in the Brain by Immunohistochemical Methods: Light, Confocal, and Electron Microscopy

Abstract

Conventional techniques to reveal the neuroanatomical distribution of type 1 cannabinoid receptor (CB₁) in the brain, at the cellular and subcellular level, are mainly represented by light, confocal, and electron microscopy. By using immunoperoxidase and immunofluorescence methods, it is possible to reveal CB₁ distribution and localization in the brain and its changes under pathological conditions. Moreover, by using electron microscopy, we can define the ultrastructural localization at the level of subcellular structures and organelles. Here, we describe immunoperoxidase, immunofluorescence, and electron microscopy protocols used to get information about CB₁ spatial distribution and localization in the brain. Preparation of reagents, resin embedding, preparation for an endogenous activity-blocking step, and background counterstaining and revelation of CB₁ by using specific labeled secondary antibodies will be presented. The methods here discussed are highly sensitive and specific multistep processes, where each step is critical to finally obtain an optimum signal.

Keywords: Biotinylated-secondary antibody; Confocal microscopy; Fluorophore-conjugated secondary antibody; Gold-conjugated antibody; Immunofluorescence; Immunoperoxidase; Transmission electron microscopy (TEM).