Saccharomyces cerevisiae nuclear and nucleolar antigen preservation for immunoelectron microscopy.
Yeast cells in general are known to be difficult to prepare for electron microscopy investigations particularly when the preservation of antigenicity is required. In this work, we compare various protocols for preparation of Saccharomyces cerevisiae cells for immunoelectron microscopy, ranging from classical chemical fixation to high-pressure freezing followed by freeze-substitution in different kinds of substitution media. Our aim was to establish a protocol giving optimal, routinely reproducible results for simultaneous retention of fine ultrastructural details and antigen immunoreactivity, with particular focus on the preservation of nuclear and nucleolar architecture. This was demonstrated by ultrastructural immunolocalization of various nucleolar (Nop1 and Nsr1), nuclear (Nsp1) and alpha-tubulin antigens. The protocol which we found to yield the best preserved Saccharomyces cerevisiae cells for both morphological and immunological studies included cryo-fixation by high-pressure freezing followed by freeze-substitution in acetone with 0.1% uranyl acetate and embedding in Lowicryl HM20. In addition, immunofluorescence detection of the antigens was performed and correlated with immunolabelling at the electron microscopy level.
Mistríková
V
V
Charles University in Prague, Institute of Cellular Biology and Pathology, Academy of Sciences of the Czech Republic, Department of Cell Biology, Czech Republic. vmist@lf1.cuni.cz
Bednár
J
J
D000096:Acetone / Q000737:chemistry; D000946:Antigens, Fungal / Q000737:chemistry*; D002466:Cell Nucleolus / Q000276:immunology* / Q000648:ultrastructure*; D002467:Cell Nucleus / Q000276:immunology* / Q000648:ultrastructure*; D005557:Formaldehyde / Q000737:chemistry; D017110:Freeze Substitution; D005976:Glutaral / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009942:Organometallic Compounds / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure*
D000946:Antigens, Fungal; D009942:Organometallic Compounds; D000096:Acetone; D005557:Formaldehyde; C005460:uranyl acetate; D005976:Glutaral