Pre-Embedding Methods for the Localization of Receptors and Ion Channels

Abstract

Pre-embedding techniques are some of the most widely used approaches in immunoelectron microscopy applied to the neurosciences, providing unexpected insights into the structure-function of neurotransmitter receptors and ion channels in the brain. These techniques involve immunoreactions with antibodies before embedding and ultrathin sectioning. As a result, they are very sensitive and show excellent resolution and good ultrastructural and antigenicity preservation. Among other advantages, pre-embedding techniques are useful for simultaneous observation of labeled tissue at the light and electron microscopic levels, as well as for quantitative analysis and 3D reconstructions. There are two main pre-embedding techniques based on the label attached to the secondary antibody: the pre-embedding immunoperoxidase method, which uses an active enzyme such as horseradish peroxidase (HRP), and the pre-embedding immunogold method, which uses a small-sized gold particle. The pre-embedding immunoperoxidase technique provides valuable information on the regional distribution of proteins of relevance for neurotransmission, while the pre-embedding immunogold technique is reliable for the localization along the neuronal surface of any specific compartment or organelle. In this chapter, we introduce pre-embedding immunoperoxidase and immunogold procedures used primarily on brain sections in both single and double labeling, and also discuss the limitations inherent to these approaches.