O estudo é apoiado por EUA Public Health Service Grant DK-081527, 072473 e DK-DK-20595 para a Universidade de Chicago Diabetes Centro de Pesquisa e Treinamento (Core modelos animais), e um presente da Fundação Família Kovler.

Parte 1: Preparação de Amostras <ol><li> Colocar um mouse sob um microscópio de dissecação, remover o pâncreas inteiro junto com o duodeno eo baço ainda ligado, e colocar os órgãos numa lâmina de vidro pré-pesados. Remove o duodeno, cortando suas tecido conjuntivo ao longo do lado onde é ligado ao pâncreas. Cortar e remover o baço e excesso de gordura sobre o pâncreas até que o pâncreas é completamente isolado no slide. (Gordura pancreática é distinguível por sua cor branca, como diferenciada da cor ligeiramente mais escura e curtidor de tecido pancreático.) </li><li> Coloque uma lamela (tampa de vidro grande 50x75mm; padrão tampa de vidro 25x75mm) sobre o pâncreas para que ele seja totalmente coberto. Use um peso (por exemplo, um livro pesado) para achatar o pâncreas entre a lâmina ea sua capa. Dependendo do peso, achatando o pâncreas leva aproximadamente cinco minutos. </li><li> Tire o peso. Se o pâncreas está bem achatado contra o slide, coloque-o em paraformaldeído 4% (PFA) a 4 ° C durante a noite. De manhã, retire as lamelas e bem expor o pâncreas inteiro para PFA durante o dia (08/06 horas). Se o órgão não está bem fixo, deixe-o permanecer no PFA mais, possivelmente durante a noite. Transferir as lâminas para o recipiente com 1% Triton x-100 em tampão fosfato salino (PBS) durante a noite. No dia seguinte, transferir os slides para o recipiente de sacarose saturada por 1-2 dias. Em seguida, transferir os slides a um recipiente de glicerol 100% até que o tecido é eliminado, o que permite a resolução ideal de sinais fluorescentes; limpeza de tecidos leva dias cerca de 1-3. (Os sinais fluorescentes podem persistir por semanas e até meses, mas de imagem deve ser realizada logo após o tecido é liberado para melhor resolução). </li><li> Loja do pâncreas em uma área escura e fria, a fim de preservar o órgão e seus sinais fluorescentes. </li></ol> Parte 2: Imagem e quantificar a área das células beta no pâncreas intacta <ol><li> Utilizando uma pinça ou luvas, pegue a deslizar para fora do recipiente com 100% de glicerol. Preparar os slides para imagens por limpando glicerol em excesso e exclusivamente a limpeza da parte de trás da tampa de vidro com etanol. </li><li> Abra o software StereoInvestigator (MicroBrightField, Williston, VT) e selecione a lente objetiva 2x para ratos imagem pancreata mais de três semanas em lâminas de vidro grande, ou a lente objetiva de 10x para os ratos imagem pancreata menos de três semanas pressionado entre duas lamínulas 25 milímetros. Visualize a imagem, colocando-o a viver de imagem (Image → Imagem Live). Selecionar um tempo de exposição que mostra claramente a GFP-tagged células-beta. (Underexposing a imagem exclui ilhotas menores e beta-células, enquanto a superexposição da imagem cria falsos sinais adicionais.) </li><li> Clique em qualquer lugar na tela para estabelecer um ponto de referência e prossiga para desenhar um contorno ao redor do pâncreas inteiro. Iniciar o processo Slice Virtual (Imagem → Adquirir Slice Virtual). Determinar e selecionar o melhor plano focal ou seja, o avião com o maior número de ilhotas visível e concentrados e células-beta no eixo Z, deslocando o botão de foco. Uma vez escolhido, StereoInvestigator captura automaticamente a imagem exibida na tela. Com o estágio motorizado, o recurso Slice Virtual seqüencialmente capta cada painel óptico como uma imagem distinta dentro do contorno desenhada (Fig. 1A) e, em seguida, combina todos eles como uma imagem incorporada (Figura 1B). Slice imagem virtual usa uma configuração de epifluorescência ou seja, campo amplo filtros que permite que a câmera para capturar todos os sinais presentes em uma certa profundidade, incluindo aqueles que não perfeitamente em foco, de modo que o Slice finais Virtual é uma imagem bidimensional integrado que não é um máximo projeção a partir da reconstrução tridimensional do pâncreas inteiro. Tempo de varredura média Virtual Slice é de 30 min por amostra, mas pode demorar até uma hora por um pâncreas grande. </li><li> Depois de salvar a imagem, começam a sua análise, abrindo a imagem com ImageJ. Quando a imagem termina de carregar e aparece na tela, abrir e executar a macro análise Virtual Slice ( <a href="https://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">Material Suplementar 1</a> ). </li><li> Siga as instruções passo-a-passo fornecidas pela macro. Eliminar os artefatos indesejados criado por dispersão de luz com a &quot;Magic Wand&quot; e &quot;Select Area&quot; ferramentas. Verifique e subtrair de fundo desnecessária da imagem. Determine o raio da maior ilhota com a &quot;Line&quot; ferramenta para que o recurso de Bola Rolando que se seguiu pode efetivamente remover a luz autofluorescência e dispersos. Teste e selecionar a mais adequada &quot;Threshold Low&quot; para capturar os sinais fluorescentes de pequenas ilhas e grupos de células-beta (&lt;3x10 M 4 2). Teste e selecionar a mais adequada &quot;Threshold High&quot; para capturar os sinais fluorescentes de ilhotas. Executar a quantificação da Slice Virtual, que irá medire produzir uma lista de área de ilhotas, perímetro, circularidade e diâmetro de Feret. </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="Figura 1A" /> Figura 1A Virtual de análise de imagem Fatia de toda a distribuição das células beta no pâncreas um adulto intacto Imagens de cada um dos painéis de óptica de um pâncreas do rato macho em 10 semanas tomadas no âmbito de um objectivo 2x. As imagens incluem toda a distribuição das ilhotas, mesmo pequenos grupos de células-beta (&lt;10 células). <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="figura 1b" /> Figura 1B análise de imagem Virtual Fatia de toda a distribuição das células beta no pâncreas um adulto intacto Uma fatia unificada virtual com o pâncreas dorsal à direita e do pâncreas ventral à esquerda. Barra de escala é de 5 mm. Resultados representante Preparação proficiente de um pâncreas para imagens Slice Virtual permitirá uma quantificação efectiva de todos os GFP-tagged células beta do pâncreas em um todo como uma imagem Slice Virtual com ilhotas claras e distintas. Imagem Slice sucesso Virtual do pâncreas todo in situ fornece os meios para examinar e quantificar as células-beta, não só a área da ilhota individual e total, mas também distribuição de tamanho de ilhotas, a análise regional, e padrões de crescimento também. Enquanto ImageJ mantém a capacidade para detectar todas as células beta-simultaneamente na análise de imagens Slice Virtual, ele também pode controlar o que as células-beta para quantificar, restringindo a análise a determinados tamanhos de ilhotas (por área ou o diâmetro), ou por localização. A análise padrão exclui artefatos por exemplo, os restos menores do que uma única célula beta-(&lt;170 M 2) e registros da área, perímetro (a distância em torno de uma área), a circularidade (um grau de circularidade, onde 1.0 representa um círculo perfeito), Feret e diâmetro (a maior distância dentro de uma área) para cada ilhota detectado. Análise Slice Virtual é capaz de aumentar a fatia de imagens virtuais através do emprego de segmentação Watershed, que distingue e separa ilhotas sobreposição em ilhotas individuais de acordo com suas formas. A análise ainda produz uma série de imagens detalhadas, o que inclui máscaras das imagens em ambos baixa e alta intensidade (Fig. 1C), um esboço de todas as ilhotas presumido, que, individualmente, sejam numerados e listados em uma tabela de informação correspondente (Fig. . 1D), ea imagem Slice originais Virtual (Figura 1B). Nota que o viés potencial técnico na escolha de valores de limiar pode distorcer a quantificação dos ilhéus, incluindo luz gratuita em torno de grandes estruturas, ou reduzindo o número total de ilhotas, os sinais fracos, especialmente a partir de pequenas partículas. <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="figura 1c" /> Figura 1C Virtual de análise de imagem Fatia de toda a distribuição das células beta no pâncreas um adulto intacto Uma máscara das partículas fluorescentes na fatia virtual. Códigos de cores: [1] azul: baixa intensidade de fluorescência; [2] verde: fluorescência intensidade intermediária, e [3] red: fluorescência de alta intensidade. <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="Figura 1D" /> Figura de análise de imagens 1D Virtual Fatia de toda a distribuição das células beta no pâncreas um adulto intacto Quantificação de ilhotas individuais / grupos de células-beta. Note que cada ilhéu (incluindo pequenos aglomerados de células-beta) é numerado, com as suas informações detalhadas em um gráfico correspondente. Quatro parâmetros foram tomadas para cada estrutura: (1) área (2); perímetro; (3) circularidade: um grau de circularidade, onde o número 1.0 representa um círculo perfeito, e (4) de diâmetro Feret: a maior distância dentro de uma área. Note que # exibe a resolução 706 análises com uma área de apenas algumas células-beta. Segmentação Watershed detecta grupos de ilhotas adjacentes, como o mostrado, e de forma adequada os distingue como ilhotas distintos (ilhotas 1554, 1555 e 1556). <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="figura 1e" /> Figura 1E Virtual de análise de imagem Fatia de toda a distribuição das células beta no pâncreas um adulto intacto 3-dimensional gráfico da análise fatia virtual, com eixos de área, perímetro e diâmetro de Feret. Cada ponto representa uma ilhota.

<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>

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A análise de imagens Slice Virtual de ilhotas e células beta no pâncreas aglomerados intacto fornece uma visão em grande escala de toda a distribuição das ilhotas. Desenvolvimentos e mudanças na distribuição total de ilhotas ao longo do tempo pode assim ser estudados e comparados. Um exemplo é demonstrado em nossa análise de formação de ilhotas pancreáticas (1). Uma análise abrangente da pancreata ratos neonatal em momentos diferentes (P1-P21) demonstrou que ilhotas são formadas por fissão. Enquanto a p...

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<td>Forceps</td>
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<td>Miltex</td>
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<td>Large glass slide</td>
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in situ quantification of pancreatic beta-cell mass in mice

Rastreamento alterações de populações de células específicas na saúde e na doença é um objetivo importante da pesquisa biomédica. O processo de proliferação de monitorização das células beta pancreáticas e crescimento das ilhotas é particularmente desafiador. Nós desenvolvemos um método para capturar a distribuição das células beta no pâncreas intacta de camundongos transgênicos com fluorescência marcadas beta-células com uma macro escrita para ImageJ (rsb.info.nih.gov / ij /). Após dissecção pancreática e limpeza de tecidos, o pâncreas inteiro é capturado como uma fatia virtual, após o que a GFP-tagged células-beta são examinados. A análise inclui a quantificação do total das células beta das ilhotas número área, e distribuição de tamanho com referência aos parâmetros e locais específicos para cada ilhota e para pequenos grupos de células-beta. Toda a distribuição das ilhotas podem ser plotados em três dimensões, e as informações a partir da distribuição do tamanho e da forma de cada ilhota permite uma comparação quantitativa e qualitativa das mudanças na área das células beta em geral num ápice.

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

O protocolo a seguir descreve o processo de dissecção pancreática para imagens fatia virtual, e posterior quantificação de todos os GFP-tagged células beta no pâncreas inteiro.

Quantificação em situ da Missa das células beta pancreáticas em camundongos

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

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Research

JoVE Journal

Biology

In situ Quantification of Pancreatic Beta-cell Mass in Mice

12.1K visualizações.  University of Chicago. O protocolo a seguir descreve o processo de dissecção pancreática para imagens fatia virtual, e posterior quantificação de todos os GFP-tagged células beta no pâncreas inteiro.

Vídeo: 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.

Transplante de ilhotas pancreáticas para a cápsula renal de ratos 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 ...

Biologia

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 &#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.

Quantificação de γH2AX Focos em resposta à radiação 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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Grampo patch e Técnicas de Perfusão para estudar canais iônicos Expresso em Xenopus Oócitos

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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.

Assistida por computador visualização de grande escala e Quantificação da Missa das ilhotas pancreáticas, distribuição de tamanho e Arquitetura

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.

Quantificação de Proteínas Usando Enriquecimento imunoafinidade Peptide Acoplado com Espectrometria de Massa

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.

Avaliação da função muscular do músculo extensor longo dos dedos<em&gt; Ex vivo</em&gt; E músculo tibial anterior<em&gt; Em situ</em&gt; Em Ratos

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.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<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.

Reação selecionada Monitoramento Espectrometria de Massa para Absolute Quantificação de Proteína

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.

Quantificação de alta resolução Celulose cristalina Acumulação em<em&gt; Arabidopsis</em&gt; Raízes para monitorar celular específico do tecido da parede Modificações

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).

Observação e Quantificação de Telomere e Sequências Repetitivas Utilizando Fluorescência<em&gt; In Situ</em&gt; Fluorescente (FISH) com PNA Sondas em<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 ...