Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes.
Despite the remarkable advances in electron microscopy, the difficulty in preserving the ultrastructural details of many plant cells is the major limitation to exploiting the full potential of this technology. The very nature of plant cells, including their hydrophobic surfaces, rigid cell walls and large vacuoles, make them recalcitrant to the efficient exchange of reagents that are crucial to preserving their fine structure. Achieving ultrastructural preservation while protecting the antigenicity of molecular epitopes has proven difficult. Here we describe two methods that provide good ultrastructural detail in plant cells while preserving the binding capacity of carbohydrate and protein epitopes. The first is a traditional, chemical-based protocol used to prepare developing grass (cereal) grain for electron microscopy and to locate carbohydrates as they are deposited using immunogold labeling. The second uses cryofixation techniques, including high-pressure freezing and freeze substitution, to prepare delicate, tip-growing pollen tubes and to locate the intracellular site of a polysaccharide synthase. Both procedures can take as long as 2 weeks to achieve results, but there is scope to fast-track some steps depending on the physical characteristics of the material being processed.
Australian Research Council Centre of Excellence in Plant Cell Walls, School of Botany, University of Melbourne, Melbourne, Victoria, Australia.
Sarah M
SM
Wilson
Antony
A
Bacic
D053000:Analytic Sample Preparation Methods / Q000379:methods*; D002241:Carbohydrates / Q000032:analysis; D000939:Epitopes / Q000032:analysis; D007150:Immunohistochemistry / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D059828:Plant Cells / Q000648:ultrastructure*; D010940:Plant Proteins / Q000032:analysis
D002241:Carbohydrates; D000939:Epitopes; D010940:Plant Proteins