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Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes

Abstract

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.

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Figure 1
Figure 2: Block trimming and sectioning images.
Figure 3: Image shows the procedure followed to antibody-label ultrathin sections on grids.
Figure 4: Images showing the uranyl acetate staining procedure.
Figure 5: Ultrathin sections through 4 DAP old barley grain central cell.
Figure 6: Ultrathin sections through 5 DAP barley grain central cell immunogold-labeled with (1,3;1,4)-β-D-glucan monoclonal antibody.
Figure 7: Micrographs show ultrastructural detail of the growing tip of high pressure–frozen N. alata pollen tubes at 1.5 and 4 h after germination.
Figure 8: High pressure–frozen N. alata pollen tubes labeled with the NaGSL1 antibody.

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Acknowledgements

This work has been supported by grants from the Australian Research Council (ARC Centre of Excellence in Plant Cell Walls), the Grains Research and Development Corporation, and the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Food Futures over many years. We acknowledge the support of many colleagues who have contributed to the refinement of these methods.

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S.M.W. conducted the experimental and data analysis work. S.M.W. and A.B. wrote the manuscript.

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Correspondence to Antony Bacic.

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Wilson, S., Bacic, A. Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nat Protoc 7, 1716–1727 (2012). https://doi.org/10.1038/nprot.2012.096

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