title: Preparation of yeast cells for live-cell imaging and indirect immunofluorescence.
abstract: In spite of their small size, the cellular morphology, structure, and protein localization of yeast cells can be successfully imaged. A detailed protocol for preparing yeast cells for live-cell imaging is described, including techniques to immobilize yeast for time-lapse microscopy. Protocols for indirect immunofluorescence are outlined, including strategies for fixation, cell wall digestion, and the use of primary and secondary antibodies conjugated to fluorescent moieties. Alternative approaches to these techniques are discussed, highlighting the advantages and disadvantages where possible. Using these protocols, investigation of yeast cell structure and protein localization will continue to yield important insights into yeast cell biology and regulation.
authors.affiliation: Department of Microbiology, Immunology and Cancer Biology, Center for Cell Signaling, University of Virginia, MSB (Hospital West), Room 7201, 800577, Charlottesville, VA, 22908, USA, lfp2n@virginia.edu.
authors.forename: Lucy F
authors.initials: LF
authors.lastname: Pemberton
mesh_terms: D003208:Concanavalin A / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D055767:Immobilized Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000648:ultrastructure; D012685:Sepharose / Q000737:chemistry
chemical_list: D055767:Immobilized Proteins; D003208:Concanavalin A; D012685:Sepharose