Abstract
Imaging cells, nuclei, and DNA in their natural spatial contexts and configurations is challenging yet required to understand the biology of genome organization, maintenance, and transmission. Live-cell imaging allows capturing dynamic changes of chromosomes in their nuclear and cellular context but lacks resolution. In contrast, imaging of fixed, spread chromosome samples provides unmatched resolution but potentially distorts configurations and spatial relations. Fixed whole-mount samples preserve chromosome configurations and cellular contexts and allow high-resolution imaging. Importantly the latter method allows simultaneous visualization of specific genomic regions (via fluorescent in situ hybridization—FISH) and proteins (via immune-localization using antibodies or tags). Here we present an advanced “whole-mount immuno-FISH” (WhoMI-FISH) method based on the published protocol by Bey Till et al. (Methods Mol Biol 1675:467–480, 2018) specifically optimized for pollen mother cells (PMCs) of Arabidopsis thaliana. It focuses on (1) specimen preparation that maintains meiocyte nuclei positions and genome organization in anthers and also on (2) simultaneous detection of specific genomic regions and meiotic proteins.
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References
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Acknowledgments
We thank the European Union (FP7-ITN 606956) and the Austrian Science Fund (SFB F34) for funding. We also thank Dr. Paul Fransz for his valuable suggestions and support during developing this protocol.
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3D video of male meiotic cells at leptotene stage. 3D video of a 39.18 × 39.18 × 10.83 μm (x, y, z) region of interest (160 nm per slice) containing male meiocytes in a syncytium. Meiocytes were stained with antibodies directed against the axial element protein ASY1 (green) and hybridized for the 45S rDNA repetitive region (red). The 3D video shows location of meiocytes within the anther and the relative positions of nuclei, nucleoli and rDNA. The stacks were processed with the Huygens Suite software (MP4 4322 kb)
3D video of male meiotic cells at zygotene stage. 3D video of a 24.99 × 24.99 × 20.79 μm (x, y, z) region of interest (160 nm per slice) containing male meiocytes in a syncytium. Meiocytes were stained with antibodies directed against the axial element protein ASY1 (green) and hybridized for the 45S rDNA repetitive region (red). The 3D video shows location of meiocytes within the anther and the relative positions of nuclei, nucleoli and rDNA. The stacks were processed with the Huygens Suite software (MP4 6944 kb)
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Sims, J., Chouaref, J., Schlögelhofer, P. (2020). Whole-Mount Immuno-FISH on Arabidopsis Meiocytes (WhoMI-FISH). In: Pradillo, M., Heckmann, S. (eds) Plant Meiosis. Methods in Molecular Biology, vol 2061. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9818-0_6
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DOI: https://doi.org/10.1007/978-1-4939-9818-0_6
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