Microscopy, TEM and particle size
Microsomal and plasma membrane enriched fractions from broccoli leaves were pelleted at 100,000 x g. For chemical fixation, pelleted vesicles were sequentially fixed with glutaraldehyde (2.5% in 100 mM phosphate buffer, 2 h at 4°C), osmium tetroxide (1% buffered, 2 h at 4°C), and tannic acid (1% in deonized water, 30 min at 22°C). The pellets were then thoroughly rinsed with water and covered with 2% low melting point agarose, then dehydrated with ethanol and epoxypropane at 22°C and embedded in Epon. Blocks were sectioned on a Leica EM UC6 ultramicrotome, collected on Formvar-coated copper grids and stained with uranyl acetate followed by lead citrate. Sections were examined using a JEOL 1011 transmission electron microscope with digital camera GATAN ORIUS SC200. For each treatment, an average of 5-10 ultrathin sections were examined.