Improved method for visualizing coated pits, microfilaments, and microtubules in cryofixed and freeze-substituted plant cells.
We have optimized the conditions for visualizing microfilaments, microtubules, and coated pits in the cortical cytoplasm of high-pressure frozen and freeze-substituted plant cells, in both tobacco root tips and onion cotyledons, individual microfilaments and the supramolecular structure of coated pits can be seen clearly in freeze-substituted samples treated with OsO4 at 40 degrees C followed by 5% uranyl acetate. Treatment with uranyl acetate alone resulted in poorly stained cytoplasmic organelles, whereas microfilaments were difficult to discern in specimen treated with OsO4 alone. The combination of a 40 degrees C OsO4 staining step followed by staining with uranyl acetate at 4 degrees C should prove useful for more detailed plant cytoskeletal/membrane studies in the future.
Murata
Takashi
T
Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Japan.
Karahara
Ichirou
I
Kozuka
Toshiaki
T
Thomas
H Giddings
HG
Staehelin
L Andrew
LA
Mineyuki
Yoshinobu
Y
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D003034:Coated Pits, Cell-Membrane / Q000648:ultrastructure*; D018548:Cotyledon / Q000648:ultrastructure; D015925:Cryopreservation; D017110:Freeze Substitution; D008854:Microscopy, Electron / Q000379:methods; D008870:Microtubules / Q000648:ultrastructure*; D019697:Onions / Q000648:ultrastructure*; D009942:Organometallic Compounds; D009993:Osmium Tetroxide; D018517:Plant Roots / Q000648:ultrastructure; D013194:Staining and Labeling / Q000379:methods; D014026:Nicotiana / Q000648:ultrastructure*
D009942:Organometallic Compounds; C005460:uranyl acetate; D009993:Osmium Tetroxide