Distribution of cortical microtubules in tobacco protoplasts. An immunofluorescence microscopic and ultrastructural study

Summary The arrangement of cortical microtubules in tobacco protoplasts is described using the following techniques: 1. Transmission electron microscopy (TEM) of thin sections of whole protoplasts, 2. TEM of negatively stained protoplast ghosts, and 3. Indirect immunofluorescence microscopy of protoplast ghosts. Ghosts were prepared by attaching freshly isolated protoplasts to glass coverslips or formvar/carbon-coated grids with poly-L-lysine and then bursting them either osmotically or by detergent treatment in the presence of a microtubule stabilizing buffer. Osmotic bursting of protoplasts yielded large pieces of plasma membrane with attached microtubules. These preparations proved very useful for measuring the density and length of cortical microtubules. Detergent treatment dissolved the plasma membrane and altered the distribution of cortical microtubules.