Postembedding immunogold cytochemistry of membrane molecules and amino acid transmitters in the central nervous system

Abstract

This chapter deals with procedures for postembedding labeling of brain sections embedded in epoxy or methacrylate resins and focuses on protocols that are based on freeze substitution of chemically fixed tissue. When optimized for the target epitope, such protocols offer a high labeling efficiency and allow simultaneous visualization of several antigens by use of different-sized gold particles. Postembedding labeling can be combined with anterograde tracing, permitting the identification of transmitter and postsynaptic receptors of identified axons. By use of tailor-made model systems, antibody selectivity can be monitored in a quantitative manner and under conditions that are representative of the immunocytochemical procedure. Such model systems also allow the generation of calibration curves for assessment of the cellular and subcellular concentration of soluble antigens. When used in conjunction with computer programs for automated acquisition and analysis of gold particles, the postembedding immunogold procedure provides an accurate representation of the cellular and subcellular distribution of proteins and small compounds such as transmitter amino acids. The present chapter provides a quantitative analysis and critical discussion of how changes in incubation parameters influence the labeling intensity. Postembedding immunogold cytochemistry stands out as a powerful technique for analysis of the chemical architecture of the central nervous system and has proved useful for investigating disease processes at the molecular level.