Preparation of yeast cells for live-cell imaging and indirect immunofluorescence

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.