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Cell-type specific chromatin analysis in whole-mount plant tissues by immunostaining


She, Wenjing; Baroux, Célia; Grossniklaus, Ueli (2017). Cell-type specific chromatin analysis in whole-mount plant tissues by immunostaining. In: Bemer, Marian; Baroux, Célia. Plant Chromatin Dynamics. New York: Springer, 443-454.

Abstract

Chromatin organization in eukaryotes is highly dynamic, playing fundamental roles in regulating diverse nuclear processes including DNA replication, transcription, and repair. Thus, the analysis of chromatin organization is of great importance for the elucidation of chromatin-mediated biological processes. Immunostaining coupled with imaging is one of the most powerful tools for chromatin analysis at the cellular level. However, in plants, it is sometimes technically challenging to apply this method due to the inaccessibility of certain cell types and/or poor penetration of the reagents into plant tissues and cells. To circumvent these limitations, we developed a highly efficient protocol enabling the analysis of chromatin modifications and nuclear organization at the single-cell level with high resolution in whole-mount plant tissues. The main procedure consists of five steps: (1) tissue fixation; (2) dissection and embedding; (3) tissue processing; (4) antibody incubation; and (5) imaging. This protocol has been simplified for the processing of multiple samples without the need for laborious tissue sectioning. Additionally, it preserves cellular morphology and chromatin organization, allowing comparative analyses of chromatin organization between different cell types or developmental stages. This protocol was successfully used for various tissues of different plant species, including Arabidopsis thaliana, Oryza sativa (rice), and Zea mays (maize). Importantly, this method is very useful to analyze poorly accessible tissues, such as female meiocytes, gametophytes, and embryos.

Abstract

Chromatin organization in eukaryotes is highly dynamic, playing fundamental roles in regulating diverse nuclear processes including DNA replication, transcription, and repair. Thus, the analysis of chromatin organization is of great importance for the elucidation of chromatin-mediated biological processes. Immunostaining coupled with imaging is one of the most powerful tools for chromatin analysis at the cellular level. However, in plants, it is sometimes technically challenging to apply this method due to the inaccessibility of certain cell types and/or poor penetration of the reagents into plant tissues and cells. To circumvent these limitations, we developed a highly efficient protocol enabling the analysis of chromatin modifications and nuclear organization at the single-cell level with high resolution in whole-mount plant tissues. The main procedure consists of five steps: (1) tissue fixation; (2) dissection and embedding; (3) tissue processing; (4) antibody incubation; and (5) imaging. This protocol has been simplified for the processing of multiple samples without the need for laborious tissue sectioning. Additionally, it preserves cellular morphology and chromatin organization, allowing comparative analyses of chromatin organization between different cell types or developmental stages. This protocol was successfully used for various tissues of different plant species, including Arabidopsis thaliana, Oryza sativa (rice), and Zea mays (maize). Importantly, this method is very useful to analyze poorly accessible tissues, such as female meiocytes, gametophytes, and embryos.

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Additional indexing

Item Type:Book Section, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Plant and Microbial Biology
07 Faculty of Science > Zurich-Basel Plant Science Center
Dewey Decimal Classification:580 Plants (Botany)
Language:English
Date:2017
Deposited On:10 Jan 2018 15:21
Last Modified:19 Feb 2018 10:14
Publisher:Springer
Series Name:Methods in Molecular Biology
Number:1675
ISSN:1064-3745
ISBN:978-1-4939-7317-0
OA Status:Closed
Publisher DOI:https://doi.org/10.1007/978-1-4939-7318-7_25
PubMed ID:29052206

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