A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae.
Yeast Saccharomyces cerevisiae has been a crucial model system for the study of a multitude of cellular processes because of its amenability to genetics, molecular biology and biochemical procedures. By contrast, the morphological analysis of this organism by immunoelectron microscopy (IEM) has remained in a primordial phase preventing researchers to routinely incorporate this technique into their investigations. Here, in addition to simple but detailed protocols to perform conventional electron microscopy (EM) on plastic embedded sections, we present a new IEM procedure adapted from the Tokuyasu method to prepare cryosections from mildly fixed cells. This novel approach allows an excellent cell preservation and the negatively stained membranes create superb contrast that leads to a unique resolution of the yeast morphology. This, plus the optimal preservation of the epitopes, permits combined localization studies with a fine resolution of protein complexes, vesicular carriers and organelles at an ultrastructural level. Importantly, we also show that this cryo-immunogold protocol can be combined with high-pressure freezing and therefore cryofixation can be employed if difficulties are encountered to immobilize a particular structure with chemical fixation. This new IEM technique will be a valuable tool for the large community of scientists using yeast as a model system, in particular for those studying membrane transport and dynamics.
Griffith
Janice
J
Department of Cell Biology, University Medical Centre Utrecht, 3584 CX Utrecht, the Netherlands.
Mari
Muriel
M
De Mazière
Ann
A
Reggiori
Fulvio
F
D003585:Cell Biology; D002462:Cell Membrane / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure; D015925:Cryopreservation; D016616:Cryoultramicrotomy / Q000379:methods*; D000939:Epitopes / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron; D015388:Organelles; D010504:Periodic Acid / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000378:metabolism* / Q000648:ultrastructure*; D014054:Tomography / Q000379:methods
D000939:Epitopes; D010504:Periodic Acid