Antibody production and immunocytochemical localization of amino acid transporters.

Abstract

This chapter details the procedural and conceptual issues that underpin the localization of high-affinity amino acid transporters by immunocytochemistry, with particular reference to the mammalian nervous system. Defining a role for transporters in any individual tissue often requires an understanding of the spatial distribution of a specific transport system. Techniques such as in situ hybridization provide clear evidence for the potential expression of a transporter by a specific cell type. In many cases however, properties such as targeting to basal or apical surfaces of, for instance, a transport epithelium or targeting to distinct plasmalemmal or cytoplasmic compartments in response to stimulation are most readily determined by the use of antibodies to detect such transporters, in intact tissues, isolated cells, or subcellular fractions of tissues. Methodologies for creating antibodies for the direct immunocytochemical localization of transporters are presented in this chapter. Issues that influence the applicability of methodologies such as postmortem delay, especially in human tissues, are also addressed.