For TEM studies of pollen from different plant species, more than one protocol for fixation and staining may be needed.
For fixation, anthers are placed in 2% glutaral-dehyde in 0.1 M phosphate buffer (pH 7.4) for 8-18 h at room temperature. After rinsing in buffer and distilled water, samples are postfixed in 2% 0s04 plus 0.8 % phosphate-buffered potassium ferrocyanide K4Fe(CN)6 3 H2O (2:1) for 8 h at 6° C. Samples are then washed in distilled water, dehydrated in 2,2-DMP followed by pure acetone, and embedded in Spurr's low-viscosity epoxy resin (SPURR 1969) or Agar low-viscosity resin.
Sections of pollen grains are routinely stained using the following methods:
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