title: Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins.
abstract: "Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.(1-4) Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.
authors.affiliation: Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Niikura
authors.affiliation: Center for Childhood Cancer and Blood Diseases, Nationwide Children's Hospital; Katsumi.Kitagawa@nationwidechildrens.org.
authors.forename: Katsumi
authors.initials: K
authors.lastname: Kitagawa
mesh_terms: D015153:Blotting, Western; D018929:Cell Culture Techniques; D002503:Centromere; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D018386:Kinetochores; D008856:Microscopy, Fluorescence / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014162:Transfection