ArticlePlant methods2023
Rapid in-solution preparation of somatic and meiotic plant cell nuclei for high-quality 3D immunoFISH and immunoFISH-GISH.
Article in Plant methods, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 4 papers.
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Who cites it
4 citing papers in PubMed, 4 citations in OpenAlex.
- Hexaploid Wheat × Barley (Triticum aestivum × Hordeum vulgare) Intergeneric Hybrids: Production and Analysis.Methods in molecular biology (Clifton, N.J.) · 2026Article
- Molecular cytogenetic mapping of Secale strictum introgressions in a perennial tetraploid rye and its diploid progenitor.PloS one · 2026Article
- A new efficient immunoprotocol to detect chromosomal/nuclear proteins along with repetitive DNA in squash preparations of formalin-fixed, long-stored root tips.Plant methods · 2025Article
- Plasticity of parental CENH3 incorporation into the centromeres in wheat × barley F1 hybrids.Frontiers in plant science · 2024Article
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Authors and funding
7 authors at 3 institutions in 1 country.
Funding
Abstract
backgroundThough multicolour labelling methods allow the routine detection of a wide range of fluorescent (immuno)probe types in molecular cytogenetics, combined applications for the simultaneous in situ detection of proteins and nucleic acids are still sporadic in plant cell biology. A major bottleneck has been the availability of high-quality plant nuclei with a balance between preservation of 3D ultrastructure and maintaining immunoreactivity. The aim of this study was to develop a quick and reliable procedure to prepare plant nuclei suitable for various combinations of immunolabelling and fluorescence in situ hybridisation methods (immunoFISH-GISH).
resultsThe mechanical removal of the cell wall and cytoplasm, instead of enzymatic degradation, resulted in a gentle, yet effective, cell permeabilisation. Rather than manually releasing the nuclei from the fixed tissues, the procedure involves in-solution cell handling throughout the fixation and the preparation steps as ended with pipetting the pure nuclei suspension onto microscope slides. The optimisation of several critical steps is described in detail. Finally, the procedure is shown to be compatible with immunolabelling, FISH and GISH as well as their simultaneous combinations.
conclusionA simple plant cell nuclei preparation procedure was developed for combined immunolabelling-in situ hybridisation methods. The main and critical elements of the procedure are: a short period of fixation, incorporation of detergents to facilitate the fixation of tissues and the penetration of probes, tissue grinding to eliminate unwanted cell components, and an optimal buffer to handle nuclei. The procedure is time efficient and is easily transferable without prior expertise.
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