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 40degreesC followed by 5% uranyl acetate. Treatment with uranyl acetate alone resulted in poorly stained cytoplasmic organelles, whereas microfilatrients were difficult to discern in specimen treated with OsO4 alone. The combination of a 40degreesC OsO4 staining step followed by staining with uranyl acetate at 4degreesC should prove useful for more detailed plant cytoskeletal/ membrane studies in the future.