Localization of neurotransmitter receptor and ion channel proteins in unfixed brains using in situ immunoblotting

The in situ immunoblotting (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 immunohistochemistry. 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. Therefore, several antibodies that are suitable for the investigation of native or denatured proteins fail to interact with their targets in fixed tissue samples used for immunohistochemical studies. The direct mechanical transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix (e.g., nitrocellulose membrane used for conventional immunoblotting) preserves the anatomical distribution patterns and structure of brain proteins. Also, the transferred proteins are readily accessible for immunochemical analysis on the surface of nitrocellulose membranes. Therefore, this method often enables the use of antibodies which do not recognize the target protein in fixed tissue samples. The histoblot method has been successfully applied to analyze 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. Compared to conventional immunoblot analysis of protein extracts from dissected brain regions, histoblots provide more accurate and direct information about the anatomical localization and expression levels of proteins. In this updated 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