Immunocytochemistry provides a powerful technique for analysis of molecular organization within cells. Using specific antibody probes together with electron microscopy (EM), the localization of a protein within individual cells can be analyzed. During the electronmicroscopic analysis of yeast cells, it is found that samples prepared for routine EM could be labeled quite successfully with affinity-purified antiserum against the membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase), and colloidal gold reagents. This chapter describes the detailed protocols of these methods. Generation of reagent antibodies of high specificity is of utmost importance for accurate immunolocalization at the EM level. The electron micrographs presented in the chapter compare several fixation protocols and resins, and the effect of these variables on immunoreactivity of HMG-CoA reductase with affinity-purified antiserum.
Bibliographical noteFunding Information:
This work was supported by grants from the California Biotechnology and Research Education Program and from the American Cancer Society to R. W. and by grants from the National Institutes of Health to J. R.