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.