This chapter describes immunocytochemical and actin decoration techniques for use with Dictyostelium amoebae at the electron microscope level of resolution that are useful for investigating the function of actin-binding proteins in vivo during morphogenetic cell movements. Techniques for localizing structural proteins in situ at high resolution in Dictyostelium, where cell behavior and morphogenetic stage can be defined with precision, are extremely valuable in defining the role of the cytoskeleton in signal transduction and morphogenetic cell movements. These techniques play a central role in establishing the in vivo functions of the structural components of the cytoskeleton. Studies using the procedures for scanning and high-voltage electron microscopy of whole-mounted cells and transmission electron microscopy of thin-sectioned cells without and with decoration of actin filaments by myosin S1 are presented. The results of the studies with IgG–gold reagents are presented. The chapter focuses on those techniques that have used for high-resolution immunocytochemistry of Dictyostelium amoebae using pre-embedding techniques. The chapter outlines the procedures for staining with gold probes could probably be used, with minor modification, with a number of other labels including ferritin, peroxidase, and avidin–biotin based methods.
ASJC Scopus subject areas
- Cell Biology