Analysis of the expression profile and regional distribution of neurotransmitter receptors and ion channels in the central nervous system using histoblots

The histoblot method is a reliable and convenient way to compare the regional distribution and expression level of different proteins in brain samples without compromising the integrity of antibody binding sites by tissue fixation, which is required for conventional immunocytochemistry. Fixation introduces covalent modifications, crosslinking and/or denaturation of proteins. These chemical modifications often alter the antibody-binding sites and cross-linked molecules may hinder the access of antibody to epitopes. The direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix offers much improved accessibility of the transferred proteins for immunochemical analysis. The histoblot method has been successfully applied to analyse the regional distribution of several neurotransmitter receptors, ion channels and other proteins in the adult and developing brains. While this technique lacks cellular resolution, it provides high sensitivity and much improved consistency compare to conventional immunohistochemical techniques, which is essential for reliable quantitative comparisons of overall expression levels of proteins in different brain regions. Compare to conventional immunoblot analysis of protein extracts from dissected brain regions, histoblots provide more accurate and direct information about the anatomical localization of proteins. In this chapter we describe the histoblot protocol we have used for the identification of quantitative changes in a wide range of neurotransmitter receptors and ion channels in various brain regions.