We thank Ueli Grossniklaus (University of Z&#252;rich) for technical and financial support. We are thankful to Valeria Gagliardini, Christof Eichenberger, Arturo Bolanos and Peter Kopf for general lab support. This research was funded by the University of Z&#252;rich, grants from the Swiss National Foundation to CB (31003A_130722) and Ueli Grossniklaus (31003A_141245 and 31003AB-126006), and the Agence Nationale de la Recherche to DG (Programme ANR-BLANC-2012).

Das Verfahren wird im Arbeitsablauf in Fig. 1 beschrieben, und die Einrichtung für die Präparation und Einbetten von Geweben sind in Figur 2 dargestellt. 1. Gewebefixierung <ol><li> Sammeln 20-30 Fruchtblätter in ein Mikrozentrifugenröhrchen mit frisch gemacht BVO Fixierungspuffer auf Eis. </li><li> Fix das Gewebe 30 min unter leichtem Schütteln bei Raumtemperatur. </li><li> Drehen Sie die Rohre, die die Fruchtblätter in Fixiermittel in einer Tischmikrozentrifuge 1 min bei 400 x g. </li><li> Entfernen Sie vorsichtig die Fixierungspuffer und 1 ml PBT, legen Sie die Röhrchen auf Eis. </li></ol> 2. Dissection and Embedding <ol><li> Bereiten fünf Eppendorf-Röhrchen mit je 200 ul einer frisch zubereiteten, 5% Acrylamid-Mix. </li><li> Vorbereitung fünf Superfrost Objektträger mit 70% Ethanol vorgereinigt und mit einem Bleistift markiert. </li><li> Tauwetter ein Aliquot von 20% und 20% APS NaPS je, auf Eis. </li><li> Nehmen Sie 4-5 Fruchtblättern mit einem Cut-Endrohr, Ortsie auf einen sauberen Objektträger, entfernen Sie das überschüssige Flüssigkeit. </li><li> Machen Längsschnitte mit einer feinen Nadel und nehmen die Fruchtblatt Wände, um Reihen von Samenanlagen wie gezeigt Abbildung 2 zu lösen, vermeiden Trocknung durch Abdecken mit PBS (nicht mehr als 10 ul). </li><li> Schnelles Hinzufügen und mischen 12 ul NaPS, 12 ul APS mit einem Aliquot von 200 ul Acrylamid-Mix. </li><li> In 30 ul der Aktiv Acrylamid auf die Samenanlagen seziert. </li><li> Deckel mit einem 20 x 20 mm Deckglas, lassen Sie polymerisieren bei Raumtemperatur 45-60 min. </li><li> Entfernen Sie das Deckglas mit einer Rasierklinge. In diesem Stadium können die Proben über Nacht bei 4 ° C in einer Glasküvette mit PBS aufbewahrt. </li></ol> 3. Gewebeverarbeitung HINWEIS: Alle Schritte außer 3.2.1, 3.2.3, 3.3.2 und 3.4.3 sind in Coplin Gläser mit 80 ml Lösung unter der chemischen Abzug bei Raumtemperatur durchgeführt. Folien sind mit einem Flachbild-Spitze Pinzette übertragen. <ol><li> Tissue Klärung undFixierung: <ol><li> Inkubation 5 min in Methanol. </li><li> Inkubation 5 min in Ethanol. </li><li> Inkubieren 30 min in Ethanol: Xylol (1:1). </li><li> Inkubation 5 min in Ethanol. </li><li> Inkubation 5 min in Methanol. </li><li> Inkubieren 15 min in Methanol und PBT (1:1) mit 2,5% Formaldehyd ergänzt. </li><li> Spülen Sie 2 x 10 min in PBT. In diesem Stadium können Folien über Nacht bei 4 ° C aufbewahrt werden </li></ol></li><li> Zellwand Verdauung: <ol><li> Tauwetter ein Aliquot der Zellwand Verdauung Mix auf Eis. </li><li> Nehmen Sie eine Folie aus der Glasküvette, lassen Sie das überschüssige Flüssigkeit, indem Sie sie senkrecht auf einem Papiertuch. </li><li> 100 l Zellwand Verdauung Mix über die Acrylamid-Pad und mit einem 23 x 46 mm Deckglas. Wiederholen Sie dies für die anderen Folien. Inkubation für 2 h bei 37 ° C in einer feuchten Kammer (in Materials beschrieben). </li><li> In PBT Waschen Sie die Folien 2 x 5 min. </li></ol></li><li> RNase A-Behandlung: <ol><li> Nehmen Sie eine Folie aus der Glasküvette, abtropfen the überschüssige Flüssigkeit wie zuvor. </li><li> Inkubieren jede Folie mit 100 ul RNAseA bei 100 ug / ml in PBS mit 1% Tween-20 für 1 h bei 37 ° C in einer feuchten Kammer. </li><li> Für 2 x 5 min in PBT Waschen Sie die Dias. </li></ol></li><li> Post-Fixierung und Permeabilisierung: <ol><li> Post-fix für 20 min in frisch gemachte PBT-F. </li><li> Spülen Sie die Objektträger für 10 min in PBT. </li><li> Durchdringbar für 2 h in PBS mit 2% Tween-20 bei 4 ° C </li><li> Spülen Sie die Folien für 2 x 5 min in PBT. </li></ol></li></ol> 4. Immunfärbung HINWEIS: Bei diesem Schritt hat die optimale Konzentration des primären Antikörpers durch Verwendung von verschiedenen Verdünnungen (1:200, 1:500, 1:1000) der Antikörper getestet werden. <ol><li> Inkubieren jede Folie mit 100 ul des primären Antikörpers in PBS mit 0,2% Tween-20 verdünnt 12-24 Stunden bei 4 ° C. </li><li> Waschen der Objektträger in PBT für 2-4 h bei Raumtemperatur unter leichtem Schütteln. </li><li> Wenden Sie dasSekundär-Antikörper 1:200 in PBS + 0,2% Tween-20 für 24 Stunden bei 4 ° C </li><li> Waschen Folien in PBT für 1 h bei Raumtemperatur unter leichtem Schütteln. </li><li> Mit 10 ug / ml Propidiumiodid in PBS für 15 min Gegenfärbung, dann spülen 15 min in PBS unter leichtem Schütteln bei Raumtemperatur. </li><li> Berg in der Anti-Fading-Flüssigkeit mountant mit 10 ug / ml Propidiumiodid ergänzt. Lassen Sie die Montagemedium aushärten 1 Stunde vor dem Erwerb durch CLSM Bilder. </li></ol> 5. Quantitative Bildgebung <ol><li> Bildaufnahme: <ol><li> Erwerben Sie hochauflösende Bilder mit CLSM, idealerweise mit einer Resonanz-Scanmodus, die bessere Erhaltung der Fluoreszenzsignale über längere Bild 23 und einen 63X Glycerin Sionslinse ermöglicht. </li><li> Testen Sie die Aufnahmeparameter wie Laserintensität, Verstärkung, Lochkamera, Voxel-Größe und Zoom-Faktor zu Beginn des Experiments, um eine Standarderwerbsverfahren zu definieren, um in ganz strikt befolgenAlle Folien für konsistente quantitative Messungen. </li><li> Überprüfen Sie, ob das Fehlen von Übersprechen zwischen Fluorochrome. Falls vorhanden, einen sequentiellen Scan eingestellt. Erwerben Sie separat und nicht gleichzeitig Übertragungs Bilder. </li><li> Führen seriell, dreidimensionale Bildaufnahme mit höchster Auflösung in x-und y-Abmessungen und mit 2x Oversampling in der z-Dimension (Nyquist-Regel). </li></ol></li><li> Bildverarbeitung: <ol><li> Rekonstruieren Serien Bilder in drei Dimensionen mit kommerziellen oder Open-Source-Software. </li><li> Definieren Konturflächen um jeden Kern (oder Zelle) von Interesse in 3D. </li><li> Quantifizierung der Fluoreszenz in jedem Kanal die Summe der Pixelintensitäten in jedem Objekt. </li><li> Exportieren Sie die Daten in Excel für statistische Auswertungen. Normalisieren Antikörper gegen Signale zB DNA-Färbung Signale. </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

In blühenden Pflanzen wird die weiblichen Fortpflanzungs Abstammung von mehreren Zellschichten einschließlich der Nucellus der Samenanlage und teguments umgeben, damit Rendering zytologische Färbung in whole-mount technisch anspruchsvoll. Hier stellen wir eine effiziente Protokoll ermöglicht die Erstellung und Bearbeitung einer großen Anzahl von Eizellen geeignet für zytologische Färbung wie Immunfärbung, DNA-Färbung und Fluoreszenz-in-situ-Hybridisierung ganz-mount. Wir erfolgreich verwendet sie für ...

In blühenden Pflanzen, die Einrichtung von Fortpflanzungslinien beginnt mit der Differenzierung von SMC, MMC weiblichen und männlichen Pollenmutterzelle. Die MMC entwickelt sich aus einer Unter nucellar epidermalen Zelle an der distalen Spitze der Samenanlage primordium und die Pollenmutterzellen entwickelt von sporenbildenden Gewebes in der Anthere locule, die tief in den Blütenorganen 1 angeordnet sind. SMCs Meiose haploide Sporen, die dann führen zu den Gametophyten bei der Mitose zu produzieren. Der weibliche Gametophyt oder Embryosack, besteht aus einer Eizelle, einer Zentralzelle, zwei und drei Synergiden antipodals. Der männliche Gametophyt oder Pollen, wird von einem vegetative Zelle und zwei Spermazellen zusammengesetzt. Während die männlichen Gametophyten bleibt ein relativ zugänglich Objekt-of-Studie wird die weiblichen Gametophyten innerhalb der Samenanlage eingebettet ist, sich selbst in der Blüte Fruchtblatt umschlossen und damit besondere Herausforderungen, um molekulare und zytologische Analysen. Kürzlich jedoch lasergestützteMikrodissektion bot eine elegante Lösung, die Transkriptom-Analysen in der MMC und weiblichen Zellen gametophytische 2-4. Neben den Kandidaten-Gen-Expressionsanalysen, zB mit RNA in situ Hybridisierung oder Reporter-Gen-Assays, zytologische Analysen ermöglicht die Untersuchung der Dynamik der endogenen zellulären Komponenten mit spezifischen zellulären direkte oder indirekte Immunfärbung. Insbesondere zytogenetische Färbung mit FISH und DNA-Färbung zusammen mit Immunfärbung von Chromatin-Veränderungen oder Chromatin-Komponenten sind zentrale Ansätze zur Chromatin-Dynamik und Atom-Organisation in Arabidopsis 5 aufzuklären. Typischerweise bringt Meiose spezifische Chromosomendynamik, die auch in pflanzlichen männlichen sucht wurde meiocytes 6,7; weitere Groß, zellspezifische Chromatin Reorganisation wahrscheinlich reflektiert dynamische epigenetische Reprogrammierung während der Pollenentwicklung 10.8 beschrieben. Im Gegensatz dazu aufgrundder relativen Unzugänglichkeit des weiblichen Gametophyten meiocyte und bleiben diese Untersuchungen technisch schwierig anzuwenden, und erfordern oft Schnitte oder manuelle Präparation und enzymatische Verdauung (siehe unten). Darüber hinaus ist die vorherrschende Mangel an optischer Klarheit ganz-mount ein Hindernis für die hochauflösende Bildgebung von Keimzellen in intakten Eizellen. Eine klassische Methode für die zytologische Analyse der Chromosomenorganisation in whole-mount Eizellen verwendet Feulgen-Färbung 13.11. Es handelt Säurehydrolyse (unter Verwendung unterchlorige Säure) der DNA, die in Protein-Denaturierung und damit zu Zerstörungen führt der Chromatinstruktur. Alternativ kann Chromosom Organisation in der weiblichen meiocytes und gametophytische Zellen mit DAPI-Färbung und Immunfärbung auf Semidünnschnitten oder seziert Embryosäcke und MMC (siehe beispielsweise 14-18) beobachtet werden. Fest steht jedoch, manuelle Präparation und Schnitte kann arbeitsintensiv sein undbehindert die qualitative und quantitative Analyse einer Vielzahl von Chromatin-Epitope. Hier bieten wir eine effiziente Protokoll, um eine große Anzahl von Arabidopsis Samenanlagen geeignet für eine Vielzahl von nachgelagerten zytologische Färbung im gesamten Montage vorzubereiten. Kurz gesagt, sind Blütenknospen in einer Fixierungslösung inkubiert werden Zeilen von Eizellen aus dem Fruchtblatt seziert und in Acrylamid eingebettet auf Folie wie für Pollen meiocytes 19,20 getan. Die eingebetteten Samenanlagen weiter geklärt und in Methanol, Ethanol und Xylol vor der Zellwand Verdauung und Permeabilisierung befestigt. Mögliche Variationen dieser Schritte werden diskutiert. Die Proben können dann für DNA-Färbung, Immunfärbung und FISH verwendet werden. Der Vorbereitungsmodus ist effizient und ermöglicht parallele Versuchsaufbau (bis zu 16 Objektträger kann an einem Tag für unterschiedliche nachgeschaltete Analyse vorbereitet werden). Die beschriebenen Behandlungen ermöglichen homogene Signale in Vollmontage und gut erhaltenen histologischen, Mobilfunk,und Kernorganisation in Keimzellen und die umliegenden nucellar Zellen, die qualitative und quantitative Vergleiche zwischen Zelltypen profitieren. Kalibriert, CLSM-basierte hochauflösende Bildgebung, gefolgt von 3-dimensionalen Rekonstruktion ermöglicht aussagekräftige quantitative Messungen von Fluoreszenzsignalen. Wir dieses Verfahren erfolgreich eingesetzt, um Chromatin-Dynamik in der Differenzierung und Entwicklung von MMC-21 weiblichen Gametophyten 22 zu analysieren; wir hier präsentieren, repräsentative Ergebnisse von Heterochromatin-Analyse, Immunfärbung Chromatin, GFP-Immunfärbung und FISH in whole-mount Samenanlagen. Wir glauben weiterhin, dass unser Protokoll wird für andere Pflanzengewebe und Arten sein.

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an efficient method for quantitative, single-cell analysis of chromatin modification and nuclear architecture in whole-mount ovules in arabidopsis

In blühenden Pflanzen ist der somatisch-to-Keimzelle Schicksal Übergang von der Spezifikation der Sporenmutterzellen (SMCs) in Blütenorgane der erwachsenen Pflanze markiert. Die weibliche SMC (Megaspore Mutterzelle, MMC) unterscheidet in der Samenanlage Primordium und Meiose. Das ausgewählte haploiden Megaspore läuft dann Mitose die vielzelligen weiblichen Gametophyten, die zu den Gameten geben wird, die Eizelle und Zentralzellen bilden zusammen mit akzessorischen Zellen. Die begrenzte Zugänglichkeit der MMC, meiocyte und weiblichen Gametophyten innerhalb der Samenanlage ist technisch anspruchsvoll für die zytologische und zytogenetische Analysen auf Einzelzellebene. Insbesondere ist eine direkte oder indirekte Immundetektion von zellulären oder Kern Epitope durch schlechte Penetration der Reagenzien innerhalb der Pflanzenzelle und Einzelzellenabbildungs ​​beeinträchtigt wird durch den Mangel an optischer Klarheit in whole-mount Gewebe demised. So eine effiziente Methode, um die nucl analysieren entwickelten wirOhr Organisation und Chromatin-Modifikation bei hoher Auflösung von Einzelzell ganz-Mount eingebettet Arabidopsis Samenanlagen. Es ist auf Dissektion und Einbettung von festen Eizellen in einer dünnen Schicht aus Acrylamid-Gel auf einen Objektträger beruht. Die Samenanlagen eingebettet werden, um chemische und enzymatische Behandlungen zur Verbesserung der Gewebe Klarheit und Durchlässigkeit für die Immunfärbung Reagenzien unterzogen. Diese Behandlungen erhalten zellulären und Chromatin-Organisation, DNA-und Protein-Epitope. Die Proben können für unterschiedliche nachgeschaltete zytologische Analysen einschließlich Chromatin Immunfärbung, Fluoreszenz in situ Hybridisierung (FISH), und DNA-Färbung für Heterochromatin Analyse verwendet werden. Die konfokale Laser-Scanning-Mikroskopie (CLSM) Bildgebung mit hoher Auflösung, gefolgt von 3D-Rekonstruktion erlaubt eine quantitative Messungen an Einzelzellauflösung.

Wir bieten eine robuste Protokoll für die großtechnische Herstellung und Verarbeitung von Arabidopsis Samenanlagen geeignet für zytologische Färbung ganz-mount. Durch die Einbettung sind die Samenanlagen behalten eine 3-dimensionale Struktur (Fig. 3). Darüber hinaus ist die Gewebeverarbeitung einschließlich der optischen Klärung ermöglicht die Abbildung subzellulärer Strukturen mit hoher Auflösung. Abbildung 4 zeigt DNA-Färbung in whole-mount Eizelle Primordia wo Het...

Watch this Scientific Journal Video about An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis at JoVE.com

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Wir bieten hier eine effiziente und zuverlässige Protokoll für die Immunfärbung, Fluoreszenz-in-situ-Hybridisierung, DNA-Färbung, gefolgt von quantitativen und hochauflösende Bildgebung in der whole-mount Arabidopsis thaliana Samenanlagen. Diese Methode wurde erfolgreich eingesetzt, um Chromatin-Modifikationen und Zellkernarchitektur zu analysieren.

Eine effiziente Methode zur quantitativen, Einzel-Zell-Analyse von Chromatin Modifikation und Reaktor Architektur in Whole-Mount-Samenanlagen in<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

an-efficient-method-for-quantitative-single-cell-analysis-chromatin

Research

JoVE Journal

Biology

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

13.1K Aufrufe.  University of Zürich.  Wir bieten hier eine effiziente und zuverlässige Protokoll für die Immunfärbung, Fluoreszenz-in-situ-Hybridisierung, DNA-Färbung, gefolgt von quantitativen und hochauflösende Bildgebung in der whole-mount Arabidopsis thaliana Samenanlagen. Diese Methode wurde erfolgreich eingesetzt, um Chromatin-Modifikationen und Zellkernarchitektur zu analysieren. 

Serie: An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Forschung

Biologie

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualisierung und Analyse von mRNA Moleküle mittels Fluoreszenz<em&gt; In Situ</em&gt; Hybridisierung in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Folienvorbereitungsmethode zur Erhaltung der dreidimensionalen Chromatinarchitektur von Hodenkeimzellen

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Mess räumliche und zeitliche Ca<sup&gt; 2 +</sup&gt; Signale in Arabidopsis-Pflanzen

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

Eine ganze Berg<em&gt; In Situ</em&gt; Hybridisierungsverfahren für die Gastropod Mollusc<em&gt; Spitzschlammschnecke</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC Protokoll Visualisiert Expression Protein bei Single Cell Resolution in Ganze Berge Vorbereitung der Haut

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualization of 3D White Adipose Tissue Structure Using Whole-mount Staining

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various techniques have been developed to study the morphology and biology of adipose tissue. However, conventional visualization methods are limited to studying the tissue in 2D sections, failing to capture the 3D architecture of the whole organ. Here we present whole-mount staining, an immunohistochemistry method that preserves intact adipose tissue morphology with minimal processing steps. Hence, the structures of adipocytes and other cellular components are maintained without distortion, achieving the most representative 3D visualization of the tissue. In addition, whole-mount staining can be combined with lineage tracing methods to determine cell fate decisions. However, this technique has some limitations to providing accurate information regarding deeper parts of adipose tissue. To overcome this limitation, whole-mount staining can be further combined with tissue clearing techniques to remove the opaqueness of tissue and allow for complete visualization of entire adipose tissue anatomy using light-sheet fluorescent microscopy. Therefore, a higher resolution and more accurate representation of adipose tissue structures can be captured with the combination of these techniques.

The focus of the present study is to demonstrate the whole-mount immunostaining and visualization technique as an ideal method for 3D imaging of adipose tissue architecture and cellular component.

Visualisierung von 3D weißen Fettgewebe Schachts ganz-Mount Färbung

Adipose tissue is an important metabolic organ with high plasticity and is responsive to environmental stimuli and nutrient status. As such, various ...

An Easy and Flexible Inoculation Method for Accurately Assessing Powdery Mildew-Infection Phenotypes of Arabidopsis and Other Plants

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in turn requires accurate determination of disease phenotypes of plants upon infection with a particular fungal pathogen. However, accurate disease phenotyping with unculturable biotrophic fungal pathogens such as powdery mildew is not easy to achieve and can be a rate-limiting step of a research project. Here, we have developed a safe, efficient, and easy-to-operate disease phenotyping system using the Arabidopsis-powdery mildew interaction as an example. This system mainly consists of three components: (i) a wooden inoculation box fitted with a removable lid mounted with a stainless steel or nylon mesh of ~50 &#181;m pores for inoculating a flat of plants with fungal spores, (ii) a transparent plastic chamber with a small front opening for minimizing spore escape while conducting inoculation inside, and (iii) a spore-dislodging and distribution method for even and effective inoculation. The protocols described here include the steps and parameters for making the inoculation box and the plastic chamber at a low cost, and a video demonstration of how to use the system to enable even inoculation with powdery mildew spores, thereby improving accuracy and reproducibility of disease phenotyping.

We present a protocol for constructing a simple spore-distribution system consisting of an inoculation box with a ~50 &#181;m mesh and a transparent plastic chamber. This can be used to evenly inoculate plants with powdery mildew spores, thereby enabling accurate and reproducible assessment of disease phenotypes of plants under study.

Eine einfache und flexible Impfmethode zur genauen Beurteilung von Phänotypen von Arabidopsis und anderen Pflanzen bei Mehltauinfektionen

Reducing crop losses due to fungal diseases requires improved understanding of the mechanisms governing plant immunity and fungal pathogenesis, which in ...