Chapter 24 Immunofluorescence Microscopy of Cilia and Flagella

Abstract

Aim of this chapter is to review the application of methods for immunofluorescence staining of motile ciliated and flagellated cells. Presented are fixations and sample preparation protocols that are routinely employed when using a previously untested antibody or studying a particular cell type for the first time. Also presented is a strategy for the simultaneous localization of two separate antigens, as well as nuclear DNA staining in the same cells. Chemical fixation, with aldehydes or treatment with cold methanol and/or acetone, is commonly employed to immobilize the antigen of interest. Because antibody probes are large molecules, preparative steps typically involve cell permeabilization to allow access to structures of interest. This step may also extract components that otherwise could contribute to unwanted levels of background labeling, possibly reducing “soluble” pools of the antigen itself. To aid in subsequent manipulations, cells are typically immobilized onto a coverglass. Highest-purity reagents and deionized water are to be used throughout. The various reagents and solutions are provided. Immunofluorescence of ciliated and flagellated cells has been described, that is, immobilization of cells and fixation—cold methanol and aldehyde—permeabilization, reduction of free aldehydes, blocking, labeling, and mounting. Under labeling, there is antibody labeling and double labeling. In double labeling it is necessary to use primary antibodies raised in different species (that is, rabbit and mouse) and the appropriate secondary antibodies conjugated to distinct fluorochromes to achieve the desired discrimination between the two primary targets. The mounting medium used is nonfluorescent, should resist evaporation, and should be formulated to reduce fading of the fluorochrome. An epifluorescent microscope, equipped with the appropriate excitation and barrier filters for each of the fluorochromes used, is required. The best result, in terms of image brightness, is obtained with the lowest magnification and highest-numerical-aperture oil immersion objective necessary to image the structure.