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An Improved Method for the Fixation, Embedding and Immunofluorescence Labeling of Resin-Embedded Plant Tissue

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Abstract

The use of antibodies as direct probes for specific macromolecules in plant cells and tissue is a well-established and extremely powerful technique and is of particular use in the post-genomics era. In this paper, we present an improved fixation, embedding, and immunofluorescence technique suitable for fixing “difficult” plant tissues such as pistils and inflorescence stems, which possess many trichomes and a thick hydrophobic cuticle. The key modification of the fixative used in the current study was the addition of a small amount of sucrose, CaCl2, and detergent into a 4% (v/v) formaldehyde and 1% (v/v) glutaraldehyde mixture without the requirement to vacuum infiltrate. The modified immunofluorescence labeling method featured an amended blocking buffer, increased number of washing steps, and the use of an aqueous mounting medium which produced intense immunolabeling signals with extremely low background. Moreover, the immunocytochemistry methodology described in this study has proven to be suitable for use on two widely studied plant species, namely, Vicia faba and Arabidopsis thaliana, and may, therefore, be applicable for use in studies of a wide range of angiosperms.

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Abbreviations

BSA:

bovine serum albumin

ECM:

extracellular matrix

FCS:

fetal calf serum

LM:

light microscopy

TEM:

transmission electron microscopy

PBS:

phosphate-buffered saline

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Acknowledgment

W. Chen was financially supported by a University of Wolverhampton PhD studentship, grant RS 328, ‘‘A molecular and structural investigation of autofertility and autosterility in Vicia faba L. (faba bean).’’ The project was associated with European Commission grant QLK5-CT-2002-02307, ‘‘Faba bean breeding for sustainable agriculture in Europe’’ (EU-Faba). We are very grateful to Dr. Paul Dupree, University of Cambridge, for supplying the antimannan antisera and to Dr. Paul Knox, University of Leeds, for supplying all of the other antibodies; to Professor F.L. Stoddard, University of Helsinki, for supervising the plant breeding experiments; to Professor W. Link, University of Göttingen, for supplying the seeds of the inbred lines; and to Professor Ian Jones, Lesley Tomkins, and the staff of the Electron Microscope Centre, University of Birmingham, for use of their TEM facilities.

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Authors and Affiliations

  1. School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton, WV1 1SB, UK

    W. Chen & T. C. Baldwin

  2. Biodiversity (Mycology and Botany), Eastern Cereals and Oilseeds Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON, K1A 0C6, Canada

    W. Chen

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  1. W. Chen
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  2. T. C. Baldwin
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Correspondence to W. Chen.

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Chen, W., Baldwin, T.C. An Improved Method for the Fixation, Embedding and Immunofluorescence Labeling of Resin-Embedded Plant Tissue. Plant Mol Biol Rep 25, 27–35 (2007). https://doi.org/10.1007/s11105-007-0004-5

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