Immunohistochemistry for ion channels and their interacting molecules: Tips for improving antibody accessibility

Abstract

Elucidating the molecular organization at synapses is essential for understanding brain function and plasticity. Immunohistochemistry is widely used as a sensitive and specifi c method in morphological studies. There are specifi c antibodies directed against receptors, ion channels, and their interacting molecules; however, it is sometimes diffi cult and ineffective to visualize synaptic proteins by conventional immunocytochemistry. This is mainly owing to the fact that the cross-linking of proteins by chemical fi xation hampers the accessibility of antibodies to antigen molecules. This is particularly true for receptors and ion channels condensed in the synaptic cleft, postsynaptic density, or trigger zone of action potentials. To overcome this problem, researchers have devised methods to improve immunohistochemical detection of proteins that are hidden or prone to be hidden in condensed molecular matrices. Of these methods, mild chemical fi xation by low paraformaldehyde concentrations or fresh frozen sections is often effective in detecting such hidden proteins. Moreover, pretreatment of sections with proteases such as pepsin is a prerequisite to detect proteins embedded in the core of the postsynaptic density, for example, NMDA-type glutamate receptors and their interacting PSD-95 protein family. In this chapter, we introduce these improving techniques for light microscopic immunohistochemistry.