Indirect Immunofluorescence for Monitoring Spindle Assembly and Disassembly in Yeast

In yeast like all eukaryotes, microtubules are a crucial element of the mitotic spindle that separates the genetic material during cell division. The assembly status and position of the mitotic spindle, as well as cytoplasmic microtubules, can be monitored easily using indirect immunofluorescence with antibodies against tubulin. A detailed protocol is described for Saccharomyces cerevisiae that involves the fixation of actively growing cells, removal of the cell wall by enzymatic digestion, post-fixation, and the application of tubulin antibodies. The use of secondary antibodies conjugated to a fluorescent moiety permit visualization of the mitotic spindle by fluorescence microscopy. Methods for the reduction of background and pre-absorption of antibodies are discussed.