L&#39;étude est soutenue par l&#39;US Public Health Service Grant DK-081527, 072473 et DK-DK-20595 à l&#39;Université de Chicago recherche sur le diabète et le Centre de Formation (Animal Core Model), et un don de la Fondation de la Famille Kovler.

Partie 1: Préparation des spécimens <ol><li> Placez une souris sous un microscope de dissection, retirer tout le pancréas avec le duodénum et la rate encore attaché, et placer les organes sur une lame de verre pré-pesés. Retirez le duodénum en coupant le tissu conjonctif le long du côté où il est lié au pancréas. Couper et enlever la rate et l&#39;excès de graisse sur le pancréas jusqu&#39;à ce que le pancréas est complètement isolée sur la diapositive. (Graisse pancréatique se distingue par sa couleur blanche, comme différencié de la couleur légèrement plus foncée et des tanneurs du tissu pancréatique.) </li><li> Placer une lamelle (couvercle en verre 50x75mm grande; norme couvercle en verre 25x75mm) sur le pancréas afin qu&#39;il soit entièrement recouvert. Utilisez un poids (par exemple, un livre lourd) pour aplatir le pancréas entre la lame et sa couverture. Selon le poids, l&#39;aplatissement du pancréas prend environ cinq minutes. </li><li> Enlever le poids. Si le pancréas est bien plaqué contre la diapositive, placez-la dans du paraformaldéhyde 4% (PFA) à 4 ° C pendant la nuit. Dans la matinée, retirer les lamelles et bien exposer le pancréas entier à l&#39;IFP au cours de la journée (6-8 heures). Si le pancréas n&#39;est pas bien fixé, lui permettent de rester dans la PFA plus, peut-être une nuit. Placer les lames dans le récipient avec 1% de Triton X-100 dans un tampon phosphate salin (PBS) pendant la nuit. Le lendemain, le transfert des diapositives sur le contenant du sucrose saturée pendant 1-2 jours. Puis transférez les diapositives dans un conteneur de glycérol 100% jusqu&#39;à ce que le tissu est effacé, ce qui permet la résolution optimale de signaux fluorescents; compensation des tissus prend environ 1-3 jours. (Les signaux fluorescents peuvent persister pendant des semaines, voire des mois, mais l&#39;imagerie doit être réalisée rapidement après que le tissu est effacé pour une résolution optimale.) </li><li> Boutique du pancréas dans un endroit sombre et frais afin de préserver l&#39;orgue et ses signaux fluorescents. </li></ol> Partie 2: Imagerie et quantification de la zone des cellules bêta dans le pancréas intacts <ol><li> L&#39;utilisation de forceps ou les mains gantées, prendre le glisser hors du conteneur avec du glycérol à 100%. Préparer la lame d&#39;imagerie par essuyer l&#39;excès de glycérol et exclusivement de nettoyage à l&#39;arrière du couvercle en verre avec de l&#39;éthanol. </li><li> Ouvrez le logiciel StereoInvestigator (MicroBrightField, Williston, VT) et sélectionnez l&#39;objectif 2x pour les souris imagerie pancréas plus de 3 semaines sur lames de verre de grande taille ou l&#39;objectif 10x pour les souris imagerie pancréas moins de 3 semaines pressée entre deux lamelles de 25mm. Visualisez l&#39;image en réglant c&#39;est de vivre l&#39;image (Image → image en direct). Sélectionner un temps d&#39;exposition qui montre clairement que la GFP-taggés cellules bêta. (Underexposing l&#39;image excluant petits ilôts et des bêta-cellules, tandis que surexposer l&#39;image crée de faux signaux supplémentaires.) </li><li> Cliquez n&#39;importe où sur l&#39;écran pour établir un point de référence et de procéder à dessiner un contour autour du pancréas entier. Initier le processus de Slice (Virtual Image → Acquérir Slice virtuel). Déterminer et sélectionner les meilleurs à savoir le plan focal, l&#39;avion avec le plus grand nombre d&#39;îlots visibles et ciblés et des bêta-cellules sur l&#39;axe Z en faisant défiler le bouton de mise. Une fois choisi, StereoInvestigator capture automatiquement l&#39;image affichée sur l&#39;écran. Avec la platine motorisée, la fonction Slice virtuel capte séquentiellement chaque panneau optique comme une image distincte dans le contour dessiné (fig. 1A), puis combine tous comme une image fusionnée (Fig. 1B). Trancher l&#39;imagerie virtuelle utilise une configuration de filtres à épifluorescence dire grand champ qui permet à l&#39;appareil photo pour capturer tous les signaux présents à une certaine profondeur, y compris ceux qui n&#39;étaient pas parfaitement au point, de sorte que la tranche finale virtuel est intégré à deux dimensions d&#39;image qui n&#39;est pas un maximum projection de la reconstruction en trois dimensions du pancréas entier. Temps moyen virtuel de balayage Slice est de 30 min par échantillon, mais peut prendre jusqu&#39;à une heure pour un pancréas grande. </li><li> Après avoir enregistré l&#39;image, commence son analyse par l&#39;ouverture de l&#39;image avec ImageJ. Lorsque l&#39;image se termine le chargement et s&#39;affiche sur l&#39;écran, ouvrez et exécutez la macro Slice Virtual analyse ( <a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">une documentation supplémentaire</a> ). </li><li> Suivez les instructions étape-par-étape fournies par la macro. Éliminer les artefacts indésirables créés par diffusion de la lumière avec le &quot;Magic Wand&quot; et &quot;Select Zone&quot; outils. Vérifiez et soustraire de fond inutiles de l&#39;image. Déterminer le rayon de la plus grande île avec la &quot;Ligne&quot; outil de sorte que la fonction de bal qui a suivi roulant peuvent éliminer efficacement la lumière d&#39;autofluorescence et dispersés. Test et sélectionner le plus approprié de «bas seuil» pour capter les signaux fluorescents de petits îlots et des grappes de cellules bêta (&lt;3x10 4 um 2). Test et sélectionner le plus approprié «seuil haut» pour capter les signaux fluorescents des îlots. Exécutez la quantification de la tranche virtuel, ce qui permettra de mesureret de produire une liste de la zone de l&#39;îlot, le périmètre, la circularité, et le diamètre de Féret. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="Figure 1a" /> Figure 1A analyse de l&#39;image virtuelle Tranche de toute la distribution des cellules bêta dans le pancréas adulte une intacts Images de panneaux individuels optiques d&#39;un pancréas de souris mâles à 10 semaines prises en vertu d&#39;un objectif 2x. Les images incluent la distribution complète des îlots, même de petits amas de cellules bêta (&lt;10 cellules). <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="figure 1b" /> Figure 1B analyse virtuelle Slice image de toute la distribution des cellules bêta dans le pancréas adulte une intacts Une tranche unique virtuel avec le pancréas dorsal sur le droit et le pancréas ventral sur la gauche. La barre d&#39;échelle est de 5 mm. Les résultats représentatifs Compétent la préparation d&#39;un pancréas pour l&#39;imagerie virtuelle Slice permettra une quantification efficace de toutes les GFP-taggés cellules bêta du pancréas dans un ensemble comme une image virtuelle Trancher avec des îlots clairs et distincts. Le succès d&#39;imagerie virtuelle Slice du pancréas entier in situ fournit les moyens d&#39;examiner et de quantifier les cellules bêta, non seulement l&#39;îlot espace individuel et au total, mais aussi la distribution de taille des îlots, des analyses régionales, et les schémas de croissance aussi bien. Alors que ImageJ maintient la capacité de détecter toutes les cellules bêta-simultanément dans l&#39;analyse des images virtuelles Slice, il peut également contrôler les cellules bêta afin de quantifier en limitant l&#39;analyse aux tailles des îlots de certains (par zone ou un diamètre), ou par emplacement. Une analyse standard exclut par exemple les artefacts, les débris de moins d&#39;un bêta-cellule unique (&lt;170 um 2) et enregistre la surface, le périmètre (la distance entourant une zone), la circularité (un degré de rondeur, où 1,0 représente un cercle parfait), et le diamètre de Féret (la plus longue distance dans une zone) pour chaque îlot détectée. Trancher l&#39;analyse virtuelle est capable d&#39;améliorer des images virtuelles en employant tranche de la segmentation des bassins versants, ce qui distingue et sépare les îlots qui se chevauchent en îlots individuels en fonction de leurs formes. L&#39;analyse produit en outre une série d&#39;images détaillées, ce qui comprend des masques de l&#39;image sous les deux de basse intensité et haute (Fig. 1C), un aperçu de tous les îlots présumés, qui sont chacun individuellement numérotés et répertoriés dans un tableau d&#39;information correspondant (Fig. . 1D), et l&#39;image virtuelle tranche d&#39;origine (Fig. 1B). Notez que le biais potentiel technique lors du choix des valeurs de seuil peuvent fausser la quantification des îlots de lumière, y compris par les grandes structures environnantes gratuite, ou en réduisant le nombre total d&#39;îlots, les signaux faibles en particulier à partir de petites particules. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="Figure 1c" /> Figure 1C analyse de l&#39;image virtuelle Tranche de toute la distribution des cellules bêta dans le pancréas adulte une intacts Un masque des particules fluorescentes dans la tranche virtuelle. Codes couleur: [1] bleu: faible intensité de fluorescence [2]; vert: la fluorescence d&#39;intensité moyenne, et [3] rouge: la fluorescence de haute intensité. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="Figure 1d" /> Figure 1D l&#39;analyse d&#39;image virtuelle Tranche de toute la distribution des cellules bêta dans le pancréas adulte une intacts Quantification des îlots individuels / groupes de cellules bêta. Notez que chaque îlot (y compris de petits amas de cellules bêta) est numéroté, avec ses informations détaillées dans un tableau correspondant. Quatre paramètres ont été pris pour chaque structure: (1) Zone; (2) du périmètre (3); circularité: un degré de rondeur, où le nombre 1.0 représente un cercle parfait, et (4) de diamètre Féret: la plus longue distance dans une zone. Notez que # 706 affiche la résolution des analyses d&#39;une superficie de seulement quelques cellules bêta. Segmentation du bassin versant détecte groupes d&#39;îlots adjacents, tels que celui illustré, et distingue de façon appropriée les îlots distincts que (îlots 1554, 1555 et 1556). <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="figure 1E" /> Figure 1E analyse de l&#39;image virtuelle Tranche de toute la distribution des cellules bêta dans le pancréas adulte une intacts 3-dimensionnel parcelle de l&#39;analyse tranches virtuelles, avec des axes de surface, périmètre, et le diamètre de Féret. Chaque point représente un îlot.

<ol>
	<li>Miller, K., Kim, A., Kilimnik, G., Moka, U., Jo, J., Periwal, V., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+formation+during+the+neonatal+development+in+mice.">Islet formation during the neonatal development in mice.</a> PloS One. 4, E7739-E7739 (2009).</li><li>Kilimnik, G., Kim, A., Jo, J., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+pancreatic+islet+distribution+in+situ+in+mice.">Quantification of pancreatic islet distribution in situ in mice.</a> Am J Physiol Endocrinol Metab. 297, E1331-E1338 (2009).</li><li>Hara, M., Wang, X., Kawamura, T., Bindokas, V. P., Dizon, R. F., Alcoser, S. Y., Magnuson, M. A., Bell, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+green+fluorescent+protein-labeled+pancreatic+beta+-cells.">Transgenic mice with green fluorescent protein-labeled pancreatic beta -cells.</a> Am J Physiol Endocrinol Metab. 284, E177-E183 (2003).</li><li>Hara, M., Dizon, R. F., Glick, B. S., Lee, C. S., Kaestner, K. H., Piston, D. W., Bindokas, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pancreatic+beta-cells+in+the+intact+pancreas.">Imaging pancreatic beta-cells in the intact pancreas.</a> Am J Physiol Endocrinol Metab. 290, E1041-E1047 (2006).</li></ol>

L&#39;analyse des images virtuelles Slice d&#39;îlots et de cellules bêta dans le pancréas grappes intactes offre une vue à grande échelle de toute la distribution des îlots. Évolution et les changements dans la distribution de l&#39;îlot au total au cours du temps peuvent ainsi être étudiées et comparées. Un tel exemple est montré dans notre analyse de la formation des îlots pancréatiques (1). Une analyse complète de pancréas des souris néonatales à des moments différents (P1-P21) a démontré que l...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14001-13</td>
<td>01n2</td>
</tr>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14072-10</td>
<td>02s5	</td>
</tr>
<tr>
<td>Large glass slide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>71862-01</td>
<td>75x51x1.2mm</td>
</tr>
<tr>
<td>Large cover glass</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc</td>
<td>260462</td>
<td>50x75mm</td>
</tr>
<tr>
<td>Glass Slide</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-550-15</td>
<td>25x75x1.0mm</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-458-5M</td>
<td>75x25mm</td>
</tr>
<tr>
<td>Cover glass</td>
<td>&nbsp;</td>
<td>Erie Scientific</td>
<td>&nbsp;</td>
<td>25mm circle</td>
</tr>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus IX-81</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>Olympus SZX12</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Investigator</td>
<td>&nbsp;</td>
<td>MicroBrightField</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MIP-GFP mice</td>
<td>&nbsp;</td>
<td>Jackson Laboratory</td>
<td>&nbsp;</td>
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in situ quantification of pancreatic beta-cell mass in mice

Traçage des changements de populations de cellules spécifiques dans la santé et la maladie est un objectif important de la recherche biomédicale. Le processus de surveillance du pancréas des cellules bêta des îlots prolifération et la croissance est particulièrement difficile. Nous avons développé une méthode pour capturer la distribution de cellules bêta dans le pancréas intact de souris transgéniques avec fluorescence taggés bêta-cellules avec une macro écrite pour ImageJ (rsb.info.nih.gov / ij /). Après dissection du pancréas et de compensation des tissus, le pancréas entier est capturé comme une tranche virtuel, après quoi le GFP-taggés bêta-cellules sont examinées. L&#39;analyse inclut la quantification du total des cellules bêta des îlots nombre domaine, et la distribution granulométrique en référence à des paramètres spécifiques et des emplacements pour chaque îlot et des petits amas de cellules bêta. La distribution complète des îlots peuvent être tracées en trois dimensions, et les informations provenant de la distribution de la taille et la forme de chaque îlot permet une comparaison quantitative et qualitative des changements dans l&#39;ensemble des cellules bêta région à un coup d&#39;œil.

Watch this Scientific Journal Video about In situ Quantification of Pancreatic Beta-cell Mass in Mice at JoVE.com

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Le protocole suivant décrit le processus de dissection pancréatique pour imagerie en coupes virtuelles, et la quantification ultérieure de tous les GFP-taggés cellules bêta dans le pancréas entier.

Dans la quantification in situ des cellules bêta pancréatiques chez des souris de masse

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

in-situ-quantification-of-pancreatic-beta-cell-mass-in-mice

Research

JoVE Journal

Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice

12.1K vues.  University of Chicago. Le protocole suivant décrit le processus de dissection pancréatique pour imagerie en coupes virtuelles, et la quantification ultérieure de tous les GFP-taggés cellules bêta dans le pancréas entier.

Vidéo: In situ Quantification of Pancreatic Beta-cell Mass in Mice

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

Transplantation d&#39;îlots pancréatiques dans la capsule du rein de souris diabétiques

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Recherche

Biologie

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 ...

Computer-assisted Large-scale Visualization and Quantification of Pancreatic Islet Mass, Size Distribution and Architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), and delta-cells (somatostatin) that are embedded in the exocrine tissues and comprise 1-2% of the entire pancreas. There is a close correlation between body and pancreas weight. Total beta-cell mass also increases proportionately to compensate for the demand for insulin in the body. What escapes this proportionate expansion is the size distribution of islets. Large animals such as humans share similar islet size distributions with mice, suggesting that this micro-organ has a certain size limit to be functional. The inability of large animal pancreata to generate proportionately larger islets is compensated for by an increase in the number of islets and by an increase in the proportion of larger islets in their overall islet size distribution. Furthermore, islets exhibit a striking plasticity in cellular composition and architecture among different species and also within the same species under various pathophysiological conditions. In the present study, we describe novel approaches for the analysis of biological image data in order to facilitate the automation of analytic processes, which allow for the analysis of large and heterogeneous data collections in the study of such dynamic biological processes and complex structures. Such studies have been hampered due to technical difficulties of unbiased sampling and generating large-scale data sets to precisely capture the complexity of biological processes of islet biology. Here we show methods to collect unbiased "representative" data within the limited availability of samples (or to minimize the sample collection) and the standard experimental settings, and to precisely analyze the complex three-dimensional structure of the islet. Computer-assisted automation allows for the collection and analysis of large-scale data sets and also assures unbiased interpretation of the data. Furthermore, the precise quantification of islet size distribution and spatial coordinates (i.e. X, Y, Z-positions) not only leads to an accurate visualization of pancreatic islet structure and composition, but also allows us to identify patterns during development and adaptation to altering conditions through mathematical modeling. The methods developed in this study are applicable to studies of many other systems and organisms as well.

Novel computer-assisted methods of large-scale procurement and analysis of immunohistochemically stained pancreatic specimens are described: (1) Virtual Slice capture of the entire section; (2) Mass analysis of large-scale data; (3) Reconstruction of 2D Virtual Slices; (4) 3D islet mapping; and (5) Mathematical analysis.

Assistée par ordinateur à grande échelle de visualisation et quantification de la messe des îlots pancréatiques, distribution de taille et d&#39;architecture

The pancreatic islet is a unique micro-organ composed of several hormone secreting endocrine cells such as beta-cells (insulin), alpha-cells (glucagon), ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

La quantification des protéines à l&#39;aide d&#39;enrichissement d&#39;immunoaffinité peptide couplé à la spectrométrie de masse

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Body movements are mainly provided by mechanical function of skeletal muscle. Skeletal muscle is composed of numerous bundles of myofibers that are sheathed by intramuscular connective tissues. Each myofiber contains many myofibrils that run longitudinally along the length of the myofiber. Myofibrils are the contractile apparatus of muscle and they are composed of repeated contractile units known as sarcomeres. A sarcomere unit contains actin and myosin filaments that are spaced by the Z discs and titin protein. Mechanical function of skeletal muscle is defined by the contractile and passive properties of muscle. The contractile properties are used to characterize the amount of force generated during muscle contraction, time of force generation and time of muscle relaxation. Any factor that affects muscle contraction (such as interaction between actin and myosin filaments, homeostasis of calcium, ATP/ADP ratio, etc.) influences the contractile properties. The passive properties refer to the elastic and viscous properties (stiffness and viscosity) of the muscle in the absence of contraction. These properties are determined by the extracellular and the intracellular structural components (such as titin) and connective tissues (mainly collagen) 1-2. The contractile and passive properties are two inseparable aspects of muscle function. For example, elbow flexion is accomplished by contraction of muscles in the anterior compartment of the upper arm and passive stretch of muscles in the posterior compartment of the upper arm. To truly understand muscle function, both contractile and passive properties should be studied.
The contractile and/or passive mechanical properties of muscle are often compromised in muscle diseases. A good example is Duchenne muscular dystrophy (DMD), a severe muscle wasting disease caused by dystrophin deficiency 3. Dystrophin is a cytoskeletal protein that stabilizes the muscle cell membrane (sarcolemma) during muscle contraction 4. In the absence of dystrophin, the sarcolemma is damaged by the shearing force generated during force transmission. This membrane tearing initiates a chain reaction which leads to muscle cell death and loss of contractile machinery. As a consequence, muscle force is reduced and dead myofibers are replaced by fibrotic tissues 5. This later change increases muscle stiffness 6. Accurate measurement of these changes provides important guide to evaluate disease progression and to determine therapeutic efficacy of novel gene/cell/pharmacological interventions. Here, we present two methods to evaluate both contractile and passive mechanical properties of the extensor digitorum longus (EDL) muscle and the contractile properties of the tibialis anterior (TA) muscle.

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Changes in limb muscle contractile and passive mechanical properties are important biomarkers for muscle diseases. This manuscript describes physiological assays to measure these properties in the murine extensor digitorum longus and tibialis anterior muscles.

Evaluation de la fonction musculaire du muscle long extenseur des orteils<em&gt; Ex vivo</em&gt; Et du jambier antérieur<em&gt; In situ</em&gt; Chez la souris

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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 ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

Selected Reaction Monitoring spectrométrie de masse pour Absolute quantification des protéines

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Résolution Quantification haut de Crystalline Cellulose Accumulation dans<em&gt; Arabidopsis</em&gt; Roots pour surveiller cellulaire spécifique d&#39;un tissu mural Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization (FISH) with PNA Probes in Caenorhabditis elegans

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase or an alternative pathway, known as alternative lengthening of telomeres (ALT)1. Recently, C. elegans has emerged as a multicellular model organism for the study of telomere and ALT2. Visualization of repetitive sequences in the genome is critical in understanding the biology of telomeres. While telomere length can be measured by telomere restriction fragment assay or quantitative PCR, these methods only provide the averaged telomere length. On the contrary, fluorescence in situ hybridization (FISH) can provide the information of the individual telomeres in cells. Here, we provide protocols and representative results of the method to determine telomere length of C. elegans by fluorescent in situ hybridization. This method provides a simple, but powerful, in situ procedure that does not cause noticeable damage to morphology. By using fluorescently labeled peptide nucleic acid (PNA) and digoxigenin-dUTP-labeled probe, we were able to visualize two different repetitive sequences: telomere repeats and template of ALT (TALT) in C. elegans embryos and gonads.

Observation and Quantification of Telomere and Repetitive Sequences Using Fluorescence In Situ Hybridization FISH with PNA Probes in Caenorhabditis elegans

We report a concise procedure of fluorescence in situ hybridization (FISH) in the gonad and embryos of Caenorhabditis elegans for observing and quantifying repetitive sequences. We successfully observed and quantified two different repetitive sequences, telomere repeats and template of alternative lengthening of telomeres (TALT).

Observation et la quantification des télomères et des séquences répétitives utilisant Fluorescence<em&gt; In Situ</em&gt; Hybridation (FISH) avec PNA Sondes en<em&gt; Caenorhabditis elegans</em

Telomere is a ribonucleoprotein structure that protects chromosomal ends from aberrant fusion and degradation. Telomere length is maintained by telomerase ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.