Chapter 24 Immunofluorescence Microscopy of Cilia and Flagella

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