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°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°C OsO₄ staining step followed by staining with uranyl acetate at 4°C should prove useful for more detailed plant cytoskeletal/membrane studies in the future.