El estudio está respaldado por EE.UU. Servicio de Salud Pública de subvención DK-081527, 072473-DK y DK-20595 de la Universidad de Chicago Diabetes Research and Training Center (Animal principales modelos), y un regalo de la Fundación de la Familia Kovler.

Parte 1: Preparación de Muestras <ol><li> Coloque un ratón bajo un microscopio de disección, extirpar el páncreas completo junto con el duodeno y el bazo están todavía atadas, y colocar los órganos en una placa de vidrio previamente pesado. Quitar el duodeno mediante la reducción de su tejido conectivo en el lado donde se une con el páncreas. Cortar y extirpar el bazo y el exceso de grasa en el páncreas hasta que el páncreas está completamente aislado en la diapositiva. (Grasa de páncreas se distingue por su color blanco, como se diferencian por el color ligeramente más oscuro y curtidor de tejido pancreático.) </li><li> Coloque un cubreobjetos (cubierta de cristal grandes 50x75mm, 25x75mm estándar cubierta de vidrio) sobre el páncreas para que sea totalmente cubierta. Utilice un peso (por ejemplo, un libro pesado) para aplanar el páncreas entre la diapositiva y su cubierta. En función del peso, el aplanamiento del páncreas tarda aproximadamente cinco minutos. </li><li> Sacar el peso. Si el páncreas está bien pegado a la diapositiva, el lugar en el 4% de paraformaldehído (PFA) a 4 ° C durante la noche. Por la mañana, quitar el cubreobjetos y así exponer el páncreas entero de PFA durante el día (6-8 horas). Si el páncreas no está bien fija, le permiten permanecer en la AFP más tiempo, posiblemente durante la noche. Transferencia de las diapositivas en el contenedor con 1% de Triton X-100 en tampón fosfato salino (PBS) durante la noche. Al día siguiente, la transferencia de las diapositivas en el contenedor de sacarosa saturada durante 1-2 días. A continuación, traslado de las diapositivas a un contenedor de 100% de glicerol hasta que el tejido se elimina, lo que permite la resolución óptima de las señales fluorescentes, en la limpieza del tejido lleva días aproximadamente 1-3. (Las señales fluorescentes pueden persistir durante semanas e incluso meses, pero las imágenes se debe realizar poco después de que el tejido está autorizado para una óptima resolución.) </li><li> Guarde el páncreas en una zona oscura y fría con el fin de preservar el órgano y sus señales fluorescentes. </li></ol> Parte 2: Imágenes y cuantificar el área de las células beta en el páncreas intacto <ol><li> El uso de pinzas o guantes, toma la diapositiva del envase con 100% de glicerol. Prepare la diapositiva de imágenes por limpiando el exceso de glicerol y exclusivamente la limpieza de la parte posterior de la cubierta de vidrio con etanol. </li><li> Abra el software StereoInvestigator (MicroBrightField, Williston, VT) y selecciona la lente del objetivo 2x para los ratones de imágenes páncreas de más de 3 semanas en portaobjetos de vidrio grande, o la lente del objetivo 10x para los ratones de imágenes páncreas menores de 3 semanas presionado entre dos cubreobjetos de 25 mm. Visualizar la imagen seleccionado para vivir de la imagen (imagen → imágenes en vivo). Seleccione un tiempo de exposición que muestra claramente la GFP-etiquetados células beta. (Subexponiendo la imagen excluye pequeños islotes y las células beta, mientras que la sobreexposición de la imagen crea más señales falsas.) </li><li> Haga clic en cualquier lugar de la pantalla para establecer un punto de referencia y proceder a dibujar un contorno alrededor de todo el páncreas. Iniciar el proceso de Cortar (imagen → Adquirir Cortar Virtual). Determinar y seleccionar el mejor ejemplo de plano focal, el avión con el mayor número de islotes visible y centrada y las células beta en el eje Z por el desplazamiento de la rueda de enfoque. Una vez elegido, StereoInvestigator captura automáticamente la imagen visualizada en la pantalla. Con la platina, la función de Cortar Virtual captura secuencial cada panel óptico como una imagen distinta en el contorno dibujado (Fig. 1A), y luego combina todos ellos como una imagen fusionada (Fig. 1B). Cortar imagen virtual utiliza una configuración de epifluorescencia es decir, filtros de campo amplio que permite a la cámara para captar todas las señales presentes en una cierta profundidad, incluidos los que no la perfección en el enfoque, por lo que el sector Virtual final es un sistema integrado de dos dimensiones de imagen que no es de un máximo proyección de la reconstrucción tridimensional de todo el páncreas. Tiempo medio de exploración Virtual Slice es de 30 minutos por muestra, pero puede tomar hasta una hora para una gran páncreas. </li><li> Después de guardar la imagen, comienza su análisis mediante la apertura de la imagen con ImageJ. Cuando termine de cargar la imagen y aparece en la pantalla, abrir y ejecutar la macro rebanada análisis virtual ( <a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">un material complementario</a> ). </li><li> Siga las instrucciones paso a paso proporcionadas por el macro. Eliminar los efectos no deseados creado por la dispersión de luz con la &quot;varita mágica&quot; y herramientas &quot;Seleccionar área&quot;. Comprobar y restar fondo innecesario de la imagen. Determinar el radio de la mayor islote con la herramienta &quot;Línea&quot; de modo que la función resultante de rodar la bola puede quitar con eficacia la luz de autofluorescencia y dispersas. Probar y seleccionar la más adecuada &quot;umbral bajo&quot; para capturar las señales fluorescentes de pequeños islotes y las agrupaciones de las células beta (&lt;3x10 4 m 2). Probar y seleccionar la más adecuada &quot;umbral alto&quot; para capturar las señales fluorescentes de los islotes. Ejecutar la cuantificación de la rebanada virtual, que mediráy elaborar una lista de zona de los islotes, el perímetro, circularidad, y el diámetro de Feret. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="Figura 1a" /> Figura 1 A Cortar Virtual de análisis de imágenes de toda la distribución de las células beta en el páncreas adulto intacto Imágenes de cada uno de los paneles ópticos de un páncreas de un ratón macho a 10 semanas adoptadas en virtud de un objetivo de 2x. Las imágenes incluyen la distribución de las islas, incluso pequeños grupos de células beta (&lt;10 células). <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="Figura 1b" /> Figura 1B Cortar Virtual de análisis de imágenes de toda la distribución de las células beta en el páncreas adulto intacto Una rebanada unificada virtual con el páncreas dorsal a la derecha y el páncreas ventral de la izquierda. Barra de escala es de 5 mm. Resultados representante Preparación competentes del páncreas para obtener imágenes de rebanada virtual va a permitir una cuantificación efectiva de todas las buenas prácticas agrarias de etiquetado de las células beta en el páncreas entero como una rebanada de imagen virtual con islotes clara y distinta. Cortar imágenes éxito virtual de todo el páncreas in situ proporciona los medios para analizar y cuantificar las células beta, no sólo la zona de islotes individuales y totales, pero también distribución del tamaño de los islotes, el análisis regional, y los patrones de crecimiento también. Mientras que ImageJ mantiene la capacidad de detectar todas las células beta de forma simultánea en el análisis de imágenes Cortar Virtual, también puede controlar que las células beta de cuantificar por restringir el análisis a los tamaños de los islotes determinado (por área o diámetro), o por ubicación. Un análisis de norma excluye por ejemplo, los artefactos, los restos más pequeños que una sola de las células beta (&lt;170 m 2) y registra el área, el perímetro (la distancia alrededor de un área), la circularidad (un grado de redondez, donde 1.0 representa un círculo perfecto), y el diámetro de Feret (la distancia más larga dentro de un área) de cada islote detectado. Cortar el análisis virtual es capaz de mejorar las imágenes virtuales corte mediante el empleo de la segmentación de cuencas, que distingue y separa los islotes se superponen en islotes individuales de acuerdo a sus formas. El análisis también produce una serie de imágenes detalladas, lo que incluye las máscaras de las imágenes en tanto baja y alta intensidad (Fig. 1C), un resumen de todas las islas supone, que son cada uno numerado individualmente y que figuran en la tabla de información correspondiente (Fig. . 1D), y la imagen original rebanada Virtual (Fig. 1B). Tenga en cuenta que el sesgo potencial técnico la hora de elegir los valores de umbral puede sesgar la cuantificación de los islotes de los alrededores, incluyendo la luz gratuita de grandes estructuras, o reduciendo el número total de los islotes, las señales débiles, especialmente a partir de pequeñas partículas. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="figura 1c" /> Figura 1 C Cortar Virtual de análisis de imágenes de toda la distribución de las células beta en el páncreas adulto intacto Una máscara de las partículas fluorescentes en la parte virtual. Códigos de colores: [1] azul: fluorescencia de baja intensidad, [2] verde: intensidad de fluorescencia intermedia, y [3] rojo: fluorescencia de alta intensidad. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="la figura 1d" /> Figura 1D análisis de la imagen virtual de pedacito de toda la distribución de las células beta en el páncreas adulto intacto La cuantificación de los islotes individuales / grupos de células beta. Tenga en cuenta que cada uno de los islotes (incluyendo pequeños grupos de células beta) está numerado, con la información detallada en un gráfico correspondiente. Cuatro parámetros se tomaron para cada estructura: (1) área, (2) perímetro, (3) la circularidad: un grado de redondez en el número 1.0 representa un círculo perfecto, y (4) Feret de diámetro: la distancia más larga dentro de un área. Tenga en cuenta que # 706 muestra la resolución de los análisis con una superficie de sólo unas pocas células beta. Segmentación de las cuencas hidrográficas detecta grupos de islotes adyacentes, como la que se muestra, y apropiadamente los distingue como islotes distintos (islotes de 1554, 1555 y 1556). <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="Figura 1E" /> Figura 1E Cortar Virtual de análisis de imágenes de toda la distribución de las células beta en el páncreas adulto intacto 3-dimensional terreno del análisis de corte virtual, con ejes de área, perímetro y el diámetro de Feret. Cada punto representa un islote.

<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>

El análisis de las imágenes rebanada virtual de los islotes y las agrupaciones de las células beta en el páncreas intacto proporciona una visión a gran escala de toda la distribución de los islotes. Evolución y los cambios en la distribución de los islotes total en el tiempo por lo tanto pueden ser estudiados y comparados. Un ejemplo se demuestra en el análisis de la formación de los islotes pancreáticos (1). Un análisis completo de páncreas ratones recién nacidos en distintos momentos (P1-P21) ha demostra...

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<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>&nbsp;</td>
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<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
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<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
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<td>Large glass slide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>71862-01</td>
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<td>Large cover glass</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc</td>
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<td>Glass Slide</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
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<td>25x75x1.0mm</td>
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<td>Cover glass</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
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<td>25mm circle</td>
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<td>Fluorescent microscope</td>
<td>&nbsp;</td>
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in situ quantification of pancreatic beta-cell mass in mice

Seguimiento de los cambios de las poblaciones de células específicas en materia de salud y la enfermedad es un objetivo importante de la investigación biomédica. El proceso de monitoreo de páncreas de las células beta del islote proliferación y el crecimiento es particularmente difícil. Hemos desarrollado un método para capturar la distribución de las células beta en el páncreas intacto de ratones transgénicos con fluorescencia marcado las células beta con una macro escrita para ImageJ (rsb.info.nih.gov / ij /). Después de la disección del páncreas y la limpieza de tejidos, todo el páncreas se captura como una rebanada virtual, después de que la GFP-etiquetados células beta son examinados. El análisis incluye la cuantificación del total de las células beta del islote número de área, y distribución de tamaño con respecto a los parámetros y lugares específicos para cada islote y para pequeños grupos de células beta. Toda la distribución de los islotes se pueden trazar en tres dimensiones, y la información de la distribución del tamaño y la forma de cada islote permite una comparación cuantitativa y cualitativa de los cambios en el total de superficie de las células beta de un vistazo.

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

El protocolo siguiente se describe el proceso de disección del páncreas para obtener imágenes de corte virtual, y la posterior cuantificación de todas las buenas prácticas agrarias de etiquetado de las células beta en el páncreas.

En la cuantificación in situ de las células beta pancreáticas misa en ratones

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 vistas.  University of Chicago. El protocolo siguiente se describe el proceso de disección del páncreas para obtener imágenes de corte virtual, y la posterior cuantificación de todas las buenas prácticas agrarias de etiquetado de las células beta en el páncreas.

Video: 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.

El trasplante de islotes pancreáticos en la cápsula renal de ratones diabéticos

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 ...

Investigación

Biología

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.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

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.

Asistida por ordenador a gran escala de visualización y cuantificación de los islotes pancreáticos, distribución de tamaño y arquitectura

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 cuantificación de proteínas mediante el enriquecimiento de inmunoafinidad péptido acoplado a espectrometría de masas

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.

Evaluación de la función muscular del músculo extensor largo digitorum<em&gt; Ex vivo</em&gt; Y tibial anterior del músculo<em&gt; In situ</em&gt; En ratones

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.

Visualización y Análisis de las moléculas de ARNm mediante fluorescencia<em&gt; In Situ</em&gt; La hibridación en<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.

Reacción seleccionada Monitoreo espectrometría de masas para Absolute Proteína cuantificación

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.

Alta resolución Cuantificación de celulosa cristalina acumulación en<em&gt; Arabidopsis</em&gt; Las raíces para monitorear la célula específica de tejido de pared Modificaciones

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).

La observación y cuantificación de los telómeros y las secuencias repetitivas usando fluorescencia<em&gt; In Situ</em&gt; La hibridación (FISH) con sondas de PNA 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.