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ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses.pdf (2.52 MB)

ImmunoFISH: Simultaneous Visualisation of Proteins and DNA Sequences Gives Insight Into Meiotic Processes in Nuclei of Grasses.

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posted on 2019-07-25, 13:38 authored by Adél Sepsi, Attila Fábián, Katalin Jäger, J. S. Heslop-Harrison, Trude Schwarzacher
ImmunoFISH is a method combining immunolabelling (IL) with fluorescent in situ hybridisation (FISH) to simultaneously detect the nuclear distribution of proteins and specific DNA sequences within chromosomes. This approach is particularly important when analysing meiotic cell division where morphogenesis of individual proteins follows stage-specific changes and is accompanied by a noticeable chromatin dynamism. The method presented here is simple and provides reliable results of high quality signal, low background staining and can be completed within 2 days following preparation. Conventional widefield epifluorescent or laser scanning microscopy can be used for high resolution and three-dimensional analysis. Fixation and preparation techniques were optimised to best preserve nuclear morphology and protein epitopes without the need for any antigen retrieval. Preparation of plant material involved short cross-linking fixation of meiotic tissues with paraformaldehyde (PFA) followed by enzyme digestion and slide-mounting. In order to avoid rapid sample degradation typical of shortly fixed plant materials, and to be able to perform IL later, slides were snap-frozen and stored at -80°C. Ultra-freezing produced a remarkable degree of structural preservation for up to 12 months, whereby sample quality was similar to that of fresh material. Harsh chemicals and sample dehydration were avoided throughout the procedure and permeability was ensured by a 0.1-0.3% detergent treatment. The ImmunoFISH method was developed specifically for studying meiosis in Triticeae, but should also be applicable to other grass and plant species.

Funding

The present work was funded by the European Union’s Seventh Framework Programme “People,” Marie Curie Actions (FP7/2007-2013) under REA grant agreement no. 625835, by the Hungarian Academy of Sciences (János Bolyai Research Scholarship, GENPROF IF 18/2012 and KEP-5/2017), and by the Hungarian Scientific Research Fund (OTKA, proposal ID 124266).

History

Citation

Frontiers in Plant Science, 2018, 9:1193.

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Old Departments Pre 01 Aug 2015/Department of Biology (Pre 01 Aug 2015)

Version

  • VoR (Version of Record)

Published in

Frontiers in Plant Science

Publisher

Frontiers Media

issn

1664-462X

Acceptance date

2018-07-25

Copyright date

2018

Available date

2019-07-25

Publisher version

https://www.frontiersin.org/articles/10.3389/fpls.2018.01193/full

Notes

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01193/full#supplementary-material

Language

en

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