Immunolabeling of Antigens in Plant Cells

Immunolabeling techniques have opened a new door to the study of plant cells, allowing visualization at the light microscope level of some components that otherwise can be seen only with electron microscopy, and rapid assessment of large numbers of cells. This chapter focuses on the use of antibodies to label plant cells that are derived from tissues. With the exception of endosperm, generative cells, and sperm cells, all plant cells have a cellulosic cell wall, which, when intact, presents a barrier to antibody entry. To make plant cell cytoplasm accessible to antibodies, the cell wall must be breached in some fashion. This often involves weakening or removing components of the cell wall enzymatically or sectioning through it. In addition, in green plants, chloroplasts display red autofluorescence. In this case, a simple solution is to choose a nonred fluorochrome for immunolabeling and to prevent red autofluorescence from reaching the microscope oculars or camera by the use of appropriate barrier filter. Commercially available wall-digesting enzymes include cellulases, hemicellulases, and pectinases. Indirect immunofluorescence labeling can be done either with a fluorochrome-labeled secondary antibody that reacts with the primary antibody, or with a biotin-labeled secondary antibody followed by fluorochrome-labeled streptavidin.