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.