Imaging Plasma Membrane Proteins by Confocal Microscopy

Confocal microscopy has become a widely used method in the study of plasma membrane proteins. A Medline search for the terms “plasma membrane” and “confocal” returns over 1300 references since 1966. Of these, over 1000 references appeared in the past 5 yr, and over 500 in the past 2 yr. The recent widespread adoption of confocal microscopy is the result of the advantages of the method, which eliminates out-of-focus light, producing sharp, high-contrast images of cells and subcellular structures even within thick sections, and the availability of increasingly powerful, sensitive, and user-friendly instruments from several manufacturers. The purpose of this chapter is to provide guidance in the use of confocal microscopy in the study of subcellular localization and trafficking of membrane proteins. Protocols for immunostaining of fixed slides and for studying protein targeting in living cells are included. An outline for the use of confocal microscopy is provided, but readers must rely on the instruction manual provided with their instruments for details.