Name: Un protocole rationalisé pour la microscopie de localisation à molécule unique dans les noyaux d’Arabidopsis (Vidéo) | JoVE Journal
Uploaded: 2026-05-08T14:05:00.000+00:00
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Description: Découvrez un protocole rationalisé de microscopie dSTORM pour visualiser l'architecture chromatinienne dans les noyaux d'Arabidopsis thaliana. Cette méthode améliore la pureté des noyaux et la cohérence du marquage.

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<ol>
	<li>Misteli, T. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+sequence:+cellular+organization+of+genome+function.">Beyond the sequence: cellular organization of genome function.</a> Cell. 128 (4), 787-800 (2007).</li><li>Rowley, M. J., Corces, V. G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizational+principles+of+3D+genome+architecture.">Organizational principles of 3D genome architecture.</a> Nat Rev Genet. 19 (12), 789-800 (2018).</li><li>Fransz, P., de Jong, J. H., Lysak, M., Castiglione, M. R., Schubert, I. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interphase+chromosomes+in+Arabidopsis+are+organized+as+well+defined+chromocenters+from+which+euchromatin+loops+emanate.">Interphase chromosomes in Arabidopsis are organized as well defined chromocenters from which euchromatin loops emanate.</a> Proc Natl Acad Sci U S A. 99 (22), 14584-14589 (2002).</li><li>Tessadori, F., Chupeau, M. C., Chupeau, Y., Knip, M., Germann, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+dissociation+and+sequential+reassembly+of+pericentric+heterochromatin+in+dedifferentiated+Arabidopsis+cells.">Large-scale dissociation and sequential reassembly of pericentric heterochromatin in dedifferentiated Arabidopsis cells.</a> J Cell Sci. 120 (7), 1200-1208 (2007).</li><li>Caballero, L., Pasternak, T., Riyazuddin, R., P&#233;rez-P&#233;rez, J. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connecting+high-resolution+3D+chromatin+maps+with+cell+division+and+cell+differentiation+at+the+root+apical+meristem.">Connecting high-resolution 3D chromatin maps with cell division and cell differentiation at the root apical meristem.</a> Plant Cell Rep. 43 (10), 232 (2024).</li><li>Pavlova, P., van Zanten, M., Snoek, B. L., de Jong, H., Fransz, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2D+morphometric+analysis+of+Arabidopsis+thaliana+nuclei+reveals+characteristic+profiles+of+different+cell+types+and+accessions.">2D morphometric analysis of Arabidopsis thaliana nuclei reveals characteristic profiles of different cell types and accessions.</a> Chromosome Res. 30 (1), 5-24 (2022).</li><li>Shao, L., Kner, P., Rego, E. H., Gustafsson, M. G. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+3D+microscopy+of+live+whole+cells+using+structured+illumination.">Super-resolution 3D microscopy of live whole cells using structured illumination.</a> Nat Methods. 8 (12), 1044-1046 (2011).</li><li>Gustafsson, M. G. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+structured-illumination+microscopy:+Wide-field+fluorescence+imaging+with+theoretically+unlimited+resolution.">Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution.</a> Proc Natl Acad Sci U S A. 102 (37), 13081-13086 (2005).</li><li>Hell, S. W., Wichmann, J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+the+diffraction+resolution+limit+by+stimulated+emission:+stimulated-emission-depletion+fluorescence+microscopy.">Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy.</a> Opt Lett, OL. 19 (11), 780-782 (1994).</li><li>Klar, T. A., Hell, S. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction+resolution+in+far-field+fluorescence+microscopy.">Subdiffraction resolution in far-field fluorescence microscopy.</a> Opt Lett, OL. 24 (14), 954-956 (1999).</li><li>Hugelier, S., Colosi, P. L., Lakadamyali, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Single-Molecule+Localization+Microscopy.">Quantitative Single-Molecule Localization Microscopy.</a> Annu Rev Biophys. 52, 139-160 (2023).</li><li>Schermelleh, L., Ferrand, A., Huser, T., Eggeling, C., Sauer, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+demystified.">Super-resolution microscopy demystified.</a> Nat Cell Biol. 21 (1), 72-84 (2019).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys J. 91 (11), 4258-4272 (2006).</li><li>Heilemann, M., van de Linde, S., Sch&#252;ttpelz, M., Kasper, R., Seefeldt, B. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subdiffraction-resolution+fluorescence+imaging+with+conventional+fluorescent+probes.">Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes.</a> Angew Chem Int Ed Engl. 47 (33), 6172-6176 (2008).</li><li>Ricci, M. A., Manzo, C., Garc&#237;a-Parajo, M. F., Lakadamyali, M., Cosma, M. P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+fibers+are+formed+by+heterogeneous+groups+of+nucleosomes+in+vivo.">Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo.</a> Cell. 160 (6), 1145-1158 (2015).</li><li>Xu, J., Ma, H., Jin, J., Uttam, S., Fu, R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+of+higher-order+chromatin+structures+at+different+epigenomic+states+in+single+mammalian+cells.">Super-resolution imaging of higher-order chromatin structures at different epigenomic states in single mammalian cells.</a> Cell Rep. 24 (4), 873-882 (2018).</li><li>Nozaki, T., Imai, R., Tanbo, M., Nagashima, R., Tamura, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+organization+of+chromatin+domains+revealed+by+super-resolution+live-cell+imaging.">Dynamic organization of chromatin domains revealed by super-resolution live-cell imaging.</a> Molecular Cell. 67 (2), 282-293.e7 (2017).</li><li>Komis, G., &#352;amajov&#225;, O., Ove&#269;ka, M., &#352;amaj, J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+in+plant+cell+imaging.">Super-resolution microscopy in plant cell imaging.</a> Trends in Plant Science. 20 (12), 834-843 (2015).</li><li>Kracik-Dyer, E., Baroux, C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+STED+Imaging+of+isolated+Arabidopsis+thaliana+nuclei.">3D STED Imaging of isolated Arabidopsis thaliana nuclei.</a> Methods Mol Biol. 2873, 263-280 (2025).</li><li>Gaifas, L., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+live+fluorescence+imaging+with+in+situ+cryoelectron+tomography+sheds+light+on+the+septation+process+in+Deinococcus+radiodurans.">Combining live fluorescence imaging with in situ cryoelectron tomography sheds light on the septation process in Deinococcus radiodurans.</a> Proceedings of the National Academy of Sciences. 122 (19), e2425047122 (2025).</li><li>de Jong, I. G., Beilharz, K., Kuipers, O. P., Veening, J. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Cell+Imaging+of+Bacillus+subtilis+and+Streptococcus+pneumoniae+using+Automated+time-lapse+microscopy.">Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated time-lapse microscopy.</a> J Vis Exp. (53), e3145 (2011).</li><li>Vauclare, P., Wulffel&#233;, J., Lacroix, F., Servant, P., Canfalonieri, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+nucleoid+remodeling+in+Deinococcus+radiodurans+is+associated+with+major+changes+in+Heat+Unstable+(HU)+protein+dynamics.">Stress-induced nucleoid remodeling in Deinococcus radiodurans is associated with major changes in Heat Unstable (HU) protein dynamics.</a> Nucleic Acids Res. 52 (11), 6406-6423 (2024).</li><li>Schubert, V. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+microscopy+&#8211;+Applications+in+plant+cell+research.">Super-resolution microscopy &#8211; Applications in plant cell research.</a> Front Plant Sci. 8, 531 (2017).</li><li>Carles, C. C., Ha, C. M., Jun, J. H., Fiume, E., Fletcher, J. C., Hennig, L., K&#246;hler, C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+Shoot+Apical+Meristem+Development.">Analyzing Shoot Apical Meristem Development.</a> Plant Developmental Biology: Methods and Protocols. , 105-129 (2010).</li><li>Chincinska, I. A. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+infiltration+in+plant+science:+old+method,+new+possibilities.">Leaf infiltration in plant science: old method, new possibilities.</a> Plant Methods. 17 (1), 83 (2021).</li><li>Tse-Kang, S. Y., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+immunity+in+C.+elegans+is+activated+by+pathogen+effector-triggered+aggregation+of+the+guard+protein+TIR-1+on+lysosome-related+organelles.">Intestinal immunity in C. elegans is activated by pathogen effector-triggered aggregation of the guard protein TIR-1 on lysosome-related organelles.</a> Immunity. 57 (10), 2280-2295.e6 (2024).</li><li>Lim, D., Ford, T. N., Chu, K. K., Mertz, J. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optically+sectioned+in+vivo+imaging+with+speckle+illumination+HiLo+microscopy.">Optically sectioned in vivo imaging with speckle illumination HiLo microscopy.</a> J Biomed Opt. 16 (1), 016014 (2011).</li><li>Culley, S., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mapping+and+minimization+of+super-resolution+optical+imaging+artifacts.">Quantitative mapping and minimization of super-resolution optical imaging artifacts.</a> Nat Methods. 15 (4), 263-266 (2018).</li><li>Xue, M., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+amino+acid+mutations+in+histone+H3.3+illuminate+the+functional+significance+of+H3K4+methylation+in+plants.">Single amino acid mutations in histone H3.3 illuminate the functional significance of H3K4 methylation in plants.</a> Nat Commun. 16 (1), 4408 (2025).</li><li>Rueden, C. T., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ2:+ImageJ+for+the+next+generation+of+scientific+image+data.">ImageJ2: ImageJ for the next generation of scientific image data.</a> BMC Bioinformatics. 18, 529 (2017).</li><li>Schindelin, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Ovesn&#253;, M., K&#345;&#237;&#382;ek, P., Borkovec, J., &#352;vindrych, Z., Hagen, G. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ThunderSTORM:+a+comprehensive+ImageJ+plugin+for+PALM+and+STORM+data+analysis+and+super-resolution+imaging.">ThunderSTORM: a comprehensive ImageJ plugin for PALM and STORM data analysis and super-resolution imaging.</a> Bioinformatics. 30 (16), 2389-2390 (2014).</li><li>Thompson, R. E., Larson, D. R., Webb, W. W. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precise+nanometer+localization+analysis+for+individual+fluorescent+probes.">Precise nanometer localization analysis for individual fluorescent probes.</a> Biophys J. 82 (5), 2775-2783 (2002).</li><li>Van Heel, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Similarity+measures+between+images.">Similarity measures between images.</a> Ultramicroscopy. 21 (1), 95-100 (1987).</li><li>Loureiro, J., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+plant+nuclei+for+estimation+of+nuclear+DNA+content:+Overview+and+best+practices.">Isolation of plant nuclei for estimation of nuclear DNA content: Overview and best practices.</a> Cytometry Part A. 99 (4), 318-327 (2021).</li><li>Deal, R. B., Henikoff, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+INTACT+method+for+cell+type-specific+gene+expression+and+chromatin+profiling+in+Arabidopsis+thaliana.">The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.</a> Nat Protoc. 6 (1), 56-68 (2011).</li><li>Duncan, S., Olsson, T. S. G., Hartley, M., Dean, C., Rosa, S. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+detecting+single+mRNA+molecules+in+Arabidopsis+thaliana.">A method for detecting single mRNA molecules in Arabidopsis thaliana.</a> Plant Methods. 12, 13 (2016).</li><li>Gutzat, R., Mittelsten Scheid, O. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+chromatin+and+RNA+from+rare+cell+types+with+fluorescence-activated+nuclear+sorting+(FANS).">Preparing chromatin and RNA from rare cell types with fluorescence-activated nuclear sorting (FANS).</a> Methods Mol Biol. 2093, 95-105 (2020).</li></ol>

<table><tbody><tr><td>Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody</td><td>Abcam</td><td>ab5095</td><td>Primary antibody used at 1/200 dilution in immunostaining buffer to visualize the active form of the polymerase (Polyclonal antibody raised in rabbit )</td></tr><tr><td>Acquisition software</td><td>Abbelight</td><td>NEO LiveImaging v2.18</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906&nbsp;&nbsp;</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C40</td><td></td></tr><tr><td>Certified Low Melting Agarose</td><td>Bio-Rad</td><td>1613111</td><td></td></tr><tr><td>Coverslips thickness 1.5H round</td><td>Marienfeld</td><td>117640</td><td>&Oslash;: 24 mm</td></tr><tr><td>D-(+)-Glucose, anhydrous, 99%</td><td>Thermo Scientific</td><td>A16828-36&nbsp;</td><td></td></tr><tr><td>Dichroic mirror</td><td>Semrock</td><td>Di03-R405/488/561/635-t1</td><td>Dichroic mirror for excitation/emission separation</td></tr><tr><td>Dulbecco&rsquo;s Phosphate Buffered Saline 10&acute;</td><td>biowest</td><td>X0515 - 500&nbsp;</td><td>Work in sterile conditions</td></tr><tr><td>Epredia Ultra Disposable Microtome Blades</td><td>FisherScientific</td><td>12191830</td><td>Low profile, single use</td></tr><tr><td>Falcon 6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate, with Lid</td><td>&nbsp;Falcon</td><td>353046</td><td></td></tr><tr><td>Formaldehyde solution 37%</td><td>Carl Roth</td><td>7398</td><td>Work under the fume hood</td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G2133</td><td></td></tr><tr><td>Glycerol</td><td>VWR</td><td>24388.295</td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 647</td><td>Thermo Scientific</td><td>A32733TR</td><td>Used at 1/200 as secondary antibody coupled to AF647</td></tr><tr><td>Hoechst 33258 solution</td><td>Sigma-Aldrich</td><td>94403</td><td>Used at 1/500 to counterstain the DNA, added to the agarose pad</td></tr><tr><td>Hydrochloric Acid Solution</td><td>Chem-Lab</td><td>CL05.0311.1000&nbsp;</td><td></td></tr><tr><td>JF549-Hoechst</td><td>Lavis lab and Open Chemistry team (Janelia)</td><td>JF549-Hoechst</td><td>Used to counterstain DNA, added to the agarose pad at 1.5 nM final concentration</td></tr><tr><td>KIMBLE Dounce tissue grinder set</td><td>Sigma-Aldrich</td><td>D8938</td><td>2 mL tube with both large (0.0030-0.0050 inch) and small (0.0005-0.0025 inch) clearance pestles</td></tr><tr><td>Lasers unit</td><td>Oxxius</td><td>L6Cc</td><td>Equipped with six lasers: 405 nm - 100 mW, 488 nm - 200 mW, 532 nm - 500 mW, 561 nm - 300 mW, 640 nm - 500 mW and 730 nm - 30 mW</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Carl Roth</td><td>HN03</td><td></td></tr><tr><td>Mercaptoethylamine (MEA)</td><td>Sigma-Aldrich</td><td>30070</td><td></td></tr><tr><td>MOPS sodium salt 98%</td><td>Fisher Scientific SAS</td><td>352590010</td><td></td></tr><tr><td>Multiband emission filter</td><td>Semrock</td><td>FF01-446/523/600/677</td><td>Multiband emission filtration to block laser scatter light</td></tr><tr><td>Nanodiamonds</td><td>Adamas Nanotechnologies</td><td>NDNV100nmHiWGA2ml</td><td>Stock 1 mg/mL</td></tr><tr><td>ORCA-Fusion Digital CMOS camera</td><td>Hamamatsu</td><td>C14440-20UP&nbsp;</td><td></td></tr><tr><td>Petri dish, 100/20 mm, PS, clear, with vents, sterile</td><td>Greiner Bio-One</td><td>664161</td><td>Used to grow plants on 1/2 MS medium</td></tr><tr><td>Petri dish, square, PS, clear, 120/120/17 mm, sterile</td><td>Greiner Bio-One</td><td>688161</td><td>Used when chopping the plant seedlings</td></tr><tr><td>pluriStrainer Mini cell strainer</td><td>pluriSelect</td><td>43-10030-50</td><td>Mesh size 30 &micro;m, suitable for 1.5 mL Eppendorf tube</td></tr><tr><td>Potassium Chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td></td></tr><tr><td>Single band emission filter</td><td>Semrock</td><td>FF01-698/70</td><td>Specific emission filter for AF647 channel</td></tr><tr><td>Single band emission filter</td><td>Chroma</td><td>ET600/50m</td><td>Specific emission filter for JF549 channel</td></tr><tr><td>Sodium chloride (99,5%)&nbsp;</td><td>Euromedex</td><td>1112-A</td><td></td></tr><tr><td>Star-Frost slides 76 x 26 mm</td><td>Knittel</td><td>&nbsp;VS112711FKB.01</td><td></td></tr><tr><td>Sterile syringe filter&oslash; 33 mm porosity 0.2 &micro;m Luer Lock cone</td><td>ClearLine</td><td>146560</td><td></td></tr><tr><td>Super-resolution system</td><td>Abbelight</td><td>SAFe360</td><td>Olympus IX83 equipped with 100x NA1.5 oil-immersion objective</td></tr><tr><td>Tri-Sodium Citrate 2H2O Gen-Apex Biomole</td><td>Prolabo</td><td>PRO-33615.268</td><td></td></tr><tr><td>Tris Base Molecular Biology Grade</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Thermo Scientific</td><td>20491</td><td></td></tr><tr><td>Triton X-100</td><td>Euromedex</td><td>2000-B</td><td></td></tr><tr><td>Tween 20</td><td>Euromedex</td><td>2001-B</td><td></td></tr><tr><td>Twinsil Speed</td><td>Picodent</td><td>13001002</td><td>Silicone sealant</td></tr><tr><td>Ultraviolet Ozone cleaning system UVOCS oven</td><td>UVOCS Inc.</td><td>T10X10</td><td>20 min exposure for coverslip cleaning&nbsp;</td></tr><tr><td>Vacuum pump</td><td>Vacuubrand</td><td>VP 100C</td><td></td></tr><tr><td>Heraeus Megafuge 16R Centrifuge</td><td>Thermo Scientific</td><td></td><td>Rotor TX-400 75003629. Centrifugation performed with Eppendorf tubes.</td></tr></tbody></table>

un protocole rationalisé pour la microscopie de localisation à molécule unique dans les noyaux d’arabidopsis

A Streamlined Protocol for Single-Molecule Localization Microscopy in Arabidopsis Nuclei

Un protocole rationalisé pour la microscopie de localisation à molécule unique dans les noyaux d’Arabidopsis

While confocal fluorescence microscopy has provided valuable insights into chromatin organization in plant nuclei, its diffraction-limited resolution ...

a-streamlined-protocol-for-single-molecule-localization-microscopy

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Analyse cellulaire spécifique de<em&gt; Arabidopsis</em&gt; Feuilles en utilisant la fluorescence par tri cellulaire activé

Nous fournissons ici un protocole efficace et fiable pour immunologique, hybridation fluorescente in situ, la coloration de l'ADN suivie quantitative, imagerie à haute résolution dans des ovules d'Arabidopsis thaliana l'ensemble du montage. Cette méthode a été utilisée avec succès pour analyser des modifications de la chromatine nucléaire et de l'architecture.

Une méthode efficace pour quantitative, analyse unicellulaire de la chromatine Modification et architecture nucléaire dans l&#39;ensemble du montage ovules dans<em&gt; Arabidopsis</em

Nous avons développé une nouvelle méthode pour exprimer conjointement plusieurs protéines chimériques fluorescentes fusion chez les plantes à surmonter les difficultés des méthodes conventionnelles. Il prend avantage d’utiliser un plasmide d’expression unique qui contient plusieurs protéines fonctionnellement indépendant exprimant des cassettes pour atteindre la co-expression de protéine.

La co-expression de multiples protéines chimériques fluorescentes Fusion de façon efficace dans les plantes

Nous présentons une méthode simple et souple pour l’exécution de vivre-imagerie fluorescente de Arabidopsis thaliana feuilles sur une longue période de temps. Nous utilisons une plante Arabidopsis transgénique exprimant un gène rapporteur fluorescent sous le contrôle d’un promoteur liées à l’immunité à titre d’exemple pour comprendre spatio-temporelle des réactions immunitaires des plantes.

Une méthode polyvalente pour le montage de Arabidopsis quitte pour l’imagerie Intravitale time-lapse

La microscopie de localisation simple molécule 3D est utilisée pour sonder les positions spatiales et les trajectoires de mouvement des protéines étiquetées fluorescentes dans les cellules bactériennes vivantes. Le protocole expérimental et d'analyse de données décrit ci-dessus détermine les comportements diffusifs répandus des protéines cytosoliques basées sur des trajectoires monomolécules mises en commun.

Microscopie de suivi à molécule unique - Outil pour déterminer les états diffusifs des molécules cytosoliques

Ici nous décrivons un protocole pour l’imagerie cellulaire vivante du cytosquelette cortical de microtubule au meristem apical de pousse et surveillant sa réponse aux changements des forces physiques.

Imagerie cellulaire en direct du cytosquelette microtubule et manipulation micromécanique de l’Arabidopsis Shoot Apical Meristem

Le mouvement des chromosomes au cours de la prophase méiotique est un phénomène mal compris qui est fondamental pour la formation des spores. Ce protocole décrit une méthode pour acquérir, analyser et quantifier les mouvements du centromère au cours de la prophase méiotique chez les méiocytes mâles d’Arabidopsis thaliana .

Caractérisation par imagerie en direct des mouvements du centromère au cours de la prophase méiotique mâle chez Arabidopsis thaliana

Nous présentons un protocole tricolore de microscopie de localisation de la chromatine à molécule unique (SMLM) qui permet une cartographie reproductible de l’euchromatine, de l’hétérochromatine et du RNAP II pour une analyse spatiale. Ce protocole permet un marquage multicolore efficace dans des environnements nucléaires denses, y compris des cibles associées à la chromatine, permettant une détection simultanée fiable.

Un protocole de microscopie de localisation multi-marquage à molécule unique pour l’étude de la chromatine dans un environnement nucléaire dense

Ce protocole d’immunolocalisation chez les méiocytes de maïs mâles maintient l’intégrité tridimensionnelle des cellules pendant la méiose. Il permet le suivi dynamique de la localisation intracellulaire de jusqu’à trois anticorps indépendants dans du matériel paraformaldéhyde conservé, en plus du DAPI, qui colore l’ADN.

Immunolocalisation tridimensionnelle chez les méiocytes de maïs

Ce procédé utilise la microscopie confocale en conjonction avec un noyau et une lignée de marqueurs de chromatine marqués fluorescentement dans Arabidopsis thaliana pour enregistrer simultanément la dynamique du noyau et de la chromatine dans les racines d’Arabidopsis et étudier les changements de ces dynamiques en réponse aux signaux développementaux et environnementaux.

Imagerie quantitative des cellules vivantes de la forme nucléaire et de la dynamique de la chromatine pendant le développement et le stress environnemental dans la racine d’Arabidopsis thaliana

Découvrez un protocole rationalisé de microscopie dSTORM pour visualiser l'architecture chromatinienne dans les noyaux d'Arabidopsis thaliana. Cette méthode améliore la pureté des noyaux et la cohérence du marquage.

Un protocole rationalisé pour la microscopie de localisation à molécule unique dans les noyaux d’Arabidopsis

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