Immunoelectron Microscopy of Chemically Fixed Developing Plant Embryos

The hydrophobic plant cell wall, large acidic central vacuole, diverse secondary compounds, intercellular airspaces, and rigid starch granules present obstacles to ultrastructure preservation and specimen sectioning. We describe modifications of fixation and embedding procedures successfully used with microbes, protists, and mammalian tissues that have overcome these obstacles. Vacuum infiltration is used to remove intercellular air rapidly replacing it with fixative and buffer preserving cellular ultrastructure while neutralizing the acidic vacuole. Vacuum infiltration of embedding resin ensures uniform embedding resin permeation allowing production of intact ultrathin sections that are stable under the electron beam and suitable for immunolabeling. The methodology described has been used for immunolocalization of non-specific lipid transfer proteins in the diverse cell types found in developing castor bean fruits but is suitable for all plant tissues.