Le soutien de l&#39;Institut australien des sciences et techniques nucléaires est reconnu. TCK a été récipiendaire du prix AINSE. Laboratoire de médecine épigénomique est soutenu par la National Health and Medical Research Council de l&#39;Australie (566 559). LM est soutenue par la recherche de Melbourne (Université de Melbourne) et biomédicale bourses d&#39;imagerie complémentaires CRC. Le soutien de Monash Micro Imaging (Drs Stephen Cody et Iska Carmichael) a été inestimable pour ce travail. MMT est reconnaissant pour l&#39;assistance d&#39;experts et de conseils du Dr Olga Sedelnikova du National Institutes of Health.

Les tissus <ol><li> Le tissu pulmonaire, embarqués dans des moules de la température de coupe optimale (PTOM) composé 4583, a été sectionné (5 um) en utilisant un Leica CM 1950 cryostat et montées sur la poly-L-lysine diapositives. </li></ol> Les échantillons congelés ont été obtenus à partir de Dr Simon Royce à l&#39;enfance Murdoch Research Institute. Le tissu pulmonaire a été obtenu à partir de souris Balb traitées / souris c et à partir d&#39;un modèle murin de la maladie chronique des voies respiratoires allergiques qui montre de nombreuses caractéristiques pathologiques de l&#39;asthme humain 6. Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le Comité d&#39;éthique animale de l&#39;Institut de recherche des enfants de Murdoch, qui adhère au Code australien des pratiques pour le soin et l&#39;utilisation des animaux de laboratoire à des fins scientifiques. <ol start="2"><li> Les articles ont été autorisés à l&#39;air sec à température ambiante pendant 1 heure. </li></ol> Immunofluorescence <ol><li> Les articles ont été fixées avec 2% (p / v) paraformaldéhyde en tampon phosphate salin (PBS) pendant 20 minutes. </li><li> Les articles ont été équilibrées avec deux lavages en PBS pendant 15 minutes chacun. </li><li> Les articles ont été traités avec de l&#39;éthanol à 70% (refroidi à -20 ° C) pendant 20 minutes, suivie de trois lavages en PBS pendant 10 minutes chacun. </li></ol> A ce point diapositives peuvent être stockés dans des conteneurs scellés à 4 ° C pendant 2 semaines. <ol start="4"><li> Les sections ont été lavées trois fois avec du PBS pendant 10 minutes chacun et ensuite bloqués avec 100 ul fraîchement préparé dans du PBS contenant de la BSA 8% dans du PBS contenant 0,5% de Tween-20 et 0,1% de Triton X-100 (PBS-TT) pendant 1 h à température ambiante . Toutes les incubations ont été effectuées dans une auge coloration humidifié. </li><li> Les sections ont été lavées avec du PBS-TT pour 5 minutes et un périmètre hydrophobe a été marquée dans le tissu avec un stylo de Pap. </li><li> L&#39;excès de tampon a été averti et 100 pi de lapin polyclonal primaire anti-γH2AX anticorps (dilution 1:500, dans 1% de BSA dans du PBS; Millipore), a été ajouté à chaque section pour une incubation de 2 heures à température ambiante. L&#39;incubation avec l&#39;anticorps primaire peut être effectuée durant la nuit à 4 ° C. Pour preuve supplémentaire, que les foyers représentent γH2AX CDB, un anticorps primaire reconnaissant une protéine de réparation ORD peuvent aussi être utilisés. Par exemple, la co-localisation des γH2AX et phospho-ATM est une combinaison couramment utilisés. </li><li> Les sections ont été lavées deux fois en PBS-TT pour 5 minutes chacun et incubées avec 100 pi d&#39;anticorps secondaire (Alexa Fluor 488 de chèvre anti-IgG de lapin dilué 1:500, dans 1% de BSA; Invitrogen) pendant 1 heure à température ambiante dans l&#39;obscurité . (Anticorps dilué a été maintenu dans l&#39;obscurité pendant toute la procédure). </li><li> Les sections ont été lavées trois fois avec du PBS-TT pour 5 minutes chacun et incubées avec 0.5mg/mL RNAse A pendant 30 min à 37 ° C. </li><li> Nucléaire contre-coloration et le montage a été réalisé avec support Vectashield contenant l&#39;iodure de propidium. </li><li> Les lames ont été scellés avec du vernis à ongles et stockés une nuit à 4 ° C dans l&#39;obscurité avant l&#39;analyse. </li></ol> Microscopie / Analyse <ol><li> Un microscope confocal Zeiss LSM510 Meta a été utilisé pour acquérir des images à l&#39;aide de la GFP standard (pour γH2AX - Alexa Fluor 488 de chèvre anti-IgG de lapin) et PI (543 nm) des lasers. </li></ol> Les images ont été acquises dans un modèle de série Z avec un pas de 0,5 um. Pendant l&#39;analyse, des avions individuels ont été déconvolué et empilés pour produire une image maximale projetée pour minimiser le chevauchement des foyers. La méthode de création d&#39;une image projetée maximale avant un comptage des particules ne doit être utilisé dans des circonstances où une section mince (par exemple 5 um) est utilisée et où le bruit de fond est négligeable. Où sections plus épaisses avec un fond important doit être analysé, une analyse plus complexes tels que compteur d&#39;objets 3D tels que décrits par Bolte et Cordelières (2006) doit être utilisé 7. <ol start="2"><li> Metamorph (Molecular Devices, USA) a été utilisé pour créer fusionné (rouge et vert) des images pour quantifier le nombre de foyers. </li></ol> Bien Metamorph peut être utilisé pour quantifier le nombre des foyers, généralement lors de l&#39;utilisation des coupes de tissus foyers sont comptés manuellement (par œil) pour plus de précision. Types de cellules spécifiques d&#39;intérêt peuvent être sélectionnés pour la quantification, par exemple, les cellules pulmonaires épithéliales. Cela peut être fait sur la base d&#39;histologie et en référence à l&#39;hématoxyline-éosine sections colorées en série.

<ol>
	<li>Rogakou, E. P., Boon, C., Redon, C., Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megabase+chromatin+domains+involved+in+DNA+double-strand+breaks+in+vivo.">Megabase chromatin domains involved in DNA double-strand breaks in vivo.</a> J. Cell Biol. 146, 905-916 (1999).</li><li>Bonner, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma+H2AX+and+cancer.">Gamma H2AX and cancer.</a> Nature Reviews Cancer. 8 (12), 957-967 (2008).</li><li>Dickey, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H2AX:+functional+roles+and+potential+applications.">H2AX: functional roles and potential applications.</a> Chromosoma. 118 (6), 683-692 (2009).</li><li>Bhogal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Late+residual+gamma-H2AX+foci+in+murine+skin+are+dose+responsive+and+predict+radiosensitivity+in+vivo.">Late residual gamma-H2AX foci in murine skin are dose responsive and predict radiosensitivity in vivo.</a> Radiat Res. 173 (1), 1-9 (2010).</li><li>Redon, C. E., Dickey, J. S., Bonner, W. M., Sedelnikova, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-H2AX+as+a+biomarker+of+DNA+damage+induced+by+ionizing+radiation+in+human+peripheral+blood+lymphocytes+and+artificial+skin.">gamma-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin.</a> Adv Space Res. 43 (8), 1171-1178 (2009).</li><li>Locke, N. R., Royce, S. G., Wainewright, J. S., Samuel, C. S., Tang, M. L., L, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+airway+remodeling+in+acute,+subacute,+and+chronic+models+of+allergic+airways+disease.">Comparison of airway remodeling in acute, subacute, and chronic models of allergic airways disease.</a> Am J Respir Cell Mol Biol. 36 (5), 625-632 (2007).</li><li>Bolte, S. &. a. m. p. ;. a. m. p., Cordelieres, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> J Microsc. 224 (Pt 3), 213-232 (2006).</li></ol>

Aux fins de cette démonstration, nous avons utilisé les tissus pulmonaires de souris Balb traitées à partir / souris c et à partir d&#39;un modèle murin de la maladie chronique des voies respiratoires allergiques. Nous quantifié les différences dans le nombre de foyers par cellule avec des moyennes légèrement plus élevée observée dans les poumons endommagés par rapport aux sections non traitées. Plus précisément, la quantification indiquée par 6,5 foyers / cellule et 9,4 foyers par / cellulaire (compta...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Bovine Serum Albumin (BSA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A7906</td>
<td>BSA (1%) is used to block any non-specific antibody binding. Primary and secondary antibodies are diluted in BSA.</td>
</tr>
<tr>
<td>PBS (without Ca2+ and Mg2+)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>17-517Q</td>
<td>&nbsp;</td>
<tr>
<td>Optimal Cutting Temperature (OCT) Compound 4583</td>
<td>&nbsp;</td>
<td>Tissue-Tek</td>
<td>4583</td>
<td>&nbsp;</td>
<tr>
<td>Superfrost Plus</td>
<td>Polysine slides</td>
<td>Menzel-Gl&auml;ser, Germany</td>
<td>SF41296SP</td>
<td>Electrostatically attracts frozen tissue sections and reduces the need for additional coatings and adhesives.</td>
</tr>
<tr>
<td>Tween 20</td>
<td>Reagent</td>
<td>Calbiochem</td>
<td>655205</td>
<td>Tween 20 (0.5%), is a detergent to permeabilise cells</td>
</tr>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>T8787</td>
<td>Triton X-100 (0.1%) used to permeabilise cells.</td>
</tr>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>158127</td>
<td>&nbsp;</td>
<tr>
<td>Anti-phospho-&gamma;H2AX (rabbit polyclonal antibody)</td>
<td>Primary Antibody</td>
<td>Upstate, USA</td>
<td>07-164</td>
<td>Dilution of primary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Alexa Fluor 488 goat anti-rabbit IgG (H+L)</td>
<td>Secondary Antibody</td>
<td>Invitrogen</td>
<td>A-11008</td>
<td>Dilution of secondary antibody (1:500), in 1% BSA.</td>
</tr>
<tr>
<td>Ethanol (70%)</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>BSPEL316</td>
<td>&nbsp;</td>
<tr>
<td>RNAse A</td>
<td>Reagent</td>
<td>Qiagen </td>
<td>19101</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield PI</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1300</td>
<td>A glycerol based mounting medium containing propidium iodide (a red nuclear stain).This medium must be used with an oil based lense that matches its refractive index. 
Note: DNA stain commonly used: 4,6-diamidino-2-phenylindole dihydrochloride (DAPI). Can only be used with microscopes with the appropriate excitation laser.</td>
</tr>
<tr>
<td>Mini PAP Pen</td>
<td>&nbsp;</td>
<td>Invitrogen, USA</td>
<td>008877</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (22x50mm)</td>
<td>&nbsp;</td>
<td>Menzel-Gl&auml;ser, Germany</td>
<td>CS2250100</td>
<td>&nbsp;</td>
<tr>
<td>Coplin Jar, glass</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>1771-OG</td>
<td>&nbsp;</td>
<tr>
<td>Staining Trough</td>
<td>&nbsp;</td>
<td>Grale Scientific P/L</td>
<td>V1991.99</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss LSM 510 Meta Confocal</td>
<td>&nbsp;</td>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CM Leica 1950 Cryostat</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Metamorph</td>
<td>Software for Imaging analysis</td>
<td>Molecular Devices, USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

quantitation of &gamma;h2ax foci in tissue samples

L&#39;ADN des cassures double brin (CDB) sont des lésions particulièrement meurtrières et génotoxiques, qui peuvent survenir soit par des processus endogènes (physiologique ou pathologique) ou par des facteurs exogènes, en particulier le rayonnement ionisant et les composés radiomimétique. La phosphorylation de l&#39;histone H2A variante, H2AX, à la sérine-139 résidus, dans le très conservé C-terminal motif SQEY, formant γH2AX, est une réponse précoce à l&#39;ADN des cassures double brin<sup&gt; 1</sup&gt;. Cet événement de phosphorylation est médiée par la phosphatidyl-inosito 3-kinase (PI3K) famille de protéines, de l&#39;ataxie télangiectasie muté (ATM), sous-unité kinase ADN-protéines catalytiques et ATM et RAD3 liés (ATR)<sup&gt; 2</sup&gt;. Globalement, les résultats d&#39;induction ORD dans la formation de foyers discrets nucléaires γH2AX qui peuvent être facilement détectés et quantifiés par immunofluorescence<sup&gt; 2</sup&gt;. Compte tenu de la spécificité et une sensibilité uniques de ce marqueur, l&#39;analyse des γH2AX foyers a conduit à une large gamme d&#39;applications dans la recherche biomédicale, en particulier en radiobiologie et en médecine nucléaire. La quantification de γH2AX foyers a été plus largement étudié dans les systèmes de culture cellulaire dans le contexte de rayonnements ionisants induit CDB. En dehors de la radiosensibilité cellulaire, immunofluorescence tests basés ont également été utilisés pour évaluer l&#39;efficacité des radiations modifier composés. En outre, γH2AX a été utilisé comme un marqueur moléculaire pour examiner l&#39;efficacité des diverses ORD induisant composés et est annoncée comme étant récemment marqueur important du vieillissement et des maladies, en particulier le cancer<sup&gt; 3</sup&gt;. En outre, les méthodes basées sur l&#39;immunofluorescence ont été adaptés à la détection de costume et la quantification des foyers γH2AX<em&gt; Ex vivo</em&gt; Et<em&gt; In vivo</em<sup&gt; 4,5</sup&gt;. Ici, nous démontrons une méthode typique d&#39;immunofluorescence pour la détection et la quantification des γH2AX foyers dans les tissus de la souris.

Watch this Scientific Journal Video about Quantitation of Î³H2AX Foci in Tissue Samples at JoVE.com

Quantitation of &amp;#947;H2AX Foci in Tissue Samples

La quantification de l&#39;ADN double-brin de brise sur la base de γH2AX foyers est devenu un outil précieux, en particulier dans la radiobiologie, pour l&#39;évaluation de la radiosensibilité des tissus et des effets des rayonnements composés de modification. Ici nous démontrons l&#39;utilisation d&#39;un test d&#39;immunofluorescence pour la quantification des γH2AX foyers dans des échantillons de tissus.

La quantification de γH2AX Foci dans les échantillons de tissus

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

quantitation-of-h2ax-foci-in-tissue-samples

Research

JoVE Journal

Biology

Quantitation of &gamma;H2AX Foci in Tissue Samples

14.3K vues.  The Alfred Medical Research and Education Precinct. La quantification de l'ADN double-brin de brise sur la base de γH2AX foyers est devenu un outil précieux, en particulier dans la radiobiologie, pour l'évaluation de la radiosensibilité des tissus et des effets des rayonnements composés de modification. Ici nous démontrons l'utilisation d'un test d'immunofluorescence pour la quantification des γH2AX foyers dans des échantillons de tissus.

Vidéo: Quantitation of &#947;H2AX Foci in Tissue Samples

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Recherche

Biologie

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolume quantification des acides nucléiques

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Method for Measuring the Activity of Deubiquitinating Enzymes in Cell Lines and Tissue Samples

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

The current protocol details a method for measuring the activity of functionally homologous deubiquitinating enzymes. Specialized probes covalently modify the enzyme and allow for detection. This method holds the potential to identify new therapeutic targets.

Méthode de mesure de l&#39;activité des enzymes de dé-ubiquitination dans des lignées cellulaires et des échantillons de tissus

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La quantification des cellules endothéliales adhésivité<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

Quantification de l&#39;expression protéique et co-localisation utilisant multiplexé Immuno-histochimie Coloration et multispectrale Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Une méthode Sectionnement améliorée pour les échantillons de tissus végétaux récalcitrantes

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

Méthodes pour classer les foyers cytoplasmiques sous forme de granulés de stress mammalien

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

La cartographie simultanée et au dosage des ribonucléotides dans l’ADN Mitochondrial humain

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

Comprehensive Workflow of Mass Spectrometry-based Shotgun Proteomics of Tissue Samples

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible datasets. However, despite the considerable technical advancements, sample preparation from biospecimens such as patient blood, CSF, and tissue still poses considerable challenges. For identifying biomarkers, tissue proteomics often provides an attractive sample source to translate the research findings from the bench to the clinic. It can reveal potential candidate biomarkers for early diagnosis of cancer and neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, etc. Tissue proteomics also yields a wealth of systemic information based on the abundance of proteins and helps to address interesting biological questions.
Quantitative proteomics analysis can be grouped into two broad categories: a label-based and a label-free approach. In the label-based approach, proteins or peptides are labeled using stable isotopes such as SILAC (stable isotope labeling with amino acids in cell culture) or by chemical tags such as ICAT (isotope-coded affinity tags), TMT (tandem mass tag) or iTRAQ (isobaric tag for relative and absolute quantitation). Label-based approaches have the advantage of more accurate quantitation of proteins and using isobaric labels, multiple samples can be analyzed in a single experiment. The label-free approach provides a cost-effective alternative to label-based approaches. Hundreds of patient samples belonging to a particular cohort can be analyzed and compared with other cohorts based on clinical features. Here, we have described an optimized quantitative proteomics workflow for tissue samples using label-free and label-based proteome profiling methods, which is crucial for applications in life sciences, especially biomarker discovery-based projects.

The described protocol provides an optimized quantitative proteomics analysis of tissue samples using two approaches: label-based and label free quantitation. Label-based approaches have the advantage of more accurate quantitation of proteins, while a label-free approach is more cost-effective and used to analyze hundreds of samples of a cohort.

Flux de travail complet de protéomique des échantillons de tissus basée sur la spectrométrie de masse

Recent advances in mass spectrometry have resulted in deep proteomic&#160;analysis along with the generation of&#160;robust and&#160;reproducible ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

Microscopie électronique à balayage par blocs série (SBF-SEM) d’échantillons de tissus biologiques

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...