دورية أكاديمية
A protocol to expand plant nuclei.
| العنوان: | A protocol to expand plant nuclei. |
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| المؤلفون: | Kubalová I; Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany., Schmidt Černohorská M; Laboratory of Adaptive Immunity, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic., Huranová M; Laboratory of Adaptive Immunity, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic., Weisshart K; Carl Zeiss Microscopy GmbH, Jena, Germany., Houben A; Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany., Schubert V; Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Seeland, Germany. Electronic address: schubertv@ipk-gatersleben.de. |
| المصدر: | Methods in cell biology [Methods Cell Biol] 2021; Vol. 161, pp. 197-216. Date of Electronic Publication: 2020 Jul 31. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: Academic Press Country of Publication: United States NLM ID: 0373334 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 0091-679X (Print) Linking ISSN: 0091679X NLM ISO Abbreviation: Methods Cell Biol Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: New York, Academic Press |
| مواضيع طبية MeSH: | Cell Nucleus* , Centromere* , Chromatin* , Plants*, Histones/genetics ; Microscopy |
| مستخلص: | The resolution achieved by conventional light microscopy is limited by light diffraction. This obstacle can be overcome either by optical super-resolution techniques or by the recently developed method to physically expand specimens, called expansion microscopy (ExM). The method utilizes polymer chemistry and the ability of a swellable polyelectrolyte hydrogel to absorb water, and thus to expand its size. The procedure was successfully applied to different species and tissue samples, mostly from the animal kingdom. Physically expanded nuclei and chromosomes in combination with specific protein labeling and super-resolution microscopy may provide new insight into the ultrastructure, dynamics, and function of plant chromatin. Here we provide a detailed protocol to expand isolated plant nuclei and visualize proteins by indirect immunolabeling. With the focus on chromatin structure, we expanded isolated barley nuclei from root tips and visualized the centromere-specific histone H3 variant CENH3. The achieved physical expansion of ~4.2 times allowed the detection of DAPI-labeled chromatin structures already by conventional wild-field (WF) microscopy with a maximal resolution of ~50-60nm. By applying structured illumination microscopy (SIM), doubling the WF resolution, chromatin structures at a resolution of ~25-35nm were observed. However, a certain distortion of the centromeric chromatin ultrastructure became obvious. (© 2021 Elsevier Inc. All rights reserved.) |
| فهرسة مساهمة: | Keywords: CENH3; Expansion microscopy; Isolated nuclei; Plant chromatin; Rabl configuration; Structured illumination microscopy |
| المشرفين على المادة: | 0 (Chromatin) 0 (Histones) |
| تواريخ الأحداث: | Date Created: 20210122 Date Completed: 20211125 Latest Revision: 20220531 |
| رمز التحديث: | 20231215 |
| DOI: | 10.1016/bs.mcb.2020.06.007 |
| PMID: | 33478690 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 0091-679X |
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| DOI: | 10.1016/bs.mcb.2020.06.007 |