支持这项研究是由美国公众卫生服务补助DK - 081527，DK - 072473和DK - 20595芝加哥糖尿病研究和培训中心（动物模型核心）的大学，Kovler家庭基金会的礼物。

第1部分:试样制备<ol><li>下解剖镜下放置鼠标，整个胰腺仍然附着与十二指肠和脾脏中删​​除，并放在一个预先称重的载玻片机关。取出沿侧切割结缔组织，它必然胰腺十二指肠。剪切和删除对胰腺的脾和多余的脂肪，直到胰腺是幻灯片上的完全隔离。 （胰腺脂肪是从胰腺组织的颜色稍深和Tanner的区别，其白色区分开来。） </li><li>将盖玻片（大盖玻璃50x75mm;标准盖玻片25x75mm）在胰腺，使其完全覆盖。使用重量（如一个沉重的书）扁平化之间的幻灯片的胰腺和它的封面。根据不同的重量，扁平化的胰腺，大约需要5分钟。 </li><li>起飞重量。如果胰腺是平坦的，对幻灯片，将在4％多聚甲醛（PFA），在4 ° C过夜。在早上，取出盖玻片，白天（6-8小时）暴露整个胰腺煤灰。如果胰腺是不固定的，允许它留在煤灰，可能一蹴而就。转让1％的幻灯片容器的Triton X - 100的磷酸盐的缓冲液（PBS）过夜。第二天，转让幻灯片饱和蔗糖的容器为1-2天。然后，将下滑到100％甘油的容器，直到组织被清除，这对荧光信号的最佳分辨率，允许组织清算大约需要1-3天。 （荧光信号可能会持续几周，甚至几​​个月，但成像应该执行后不久，该组织是为最佳分辨率清除。） </li><li>储存在阴凉的区域胰腺，以维护机关及其荧光信号。 </li></ol>第2部分:成像和量化完整的胰腺β细胞的面积<ol><li>使用镊子或戴手套的手，容器的滑出100％的甘油。擦去多余的甘油和专门用乙醇清洗玻璃盖的背面，准备用于成像的幻灯片。 </li><li>打开StereoInvestigator软件（MicroBrightField，威利斯顿，VT），并选择2X物镜成像小鼠胰腺大型玻片3周以上，或10倍的物镜成像小鼠胰腺年龄小于3周两25毫米盖玻片压。可视化设置实时图像（图像→现场图像）图像。选择一个曝光时间，清楚地表明GFP标记的β细胞。 （曝光不足的图像排除较小的胰岛β细胞，而过度曝光的图像创建额外的虚假信号。） </li><li>点击屏幕上的任何地方建立一个基准点，并着手拟订一个围绕整个胰腺的轮廓。启动虚拟切片的过程（图像→获取虚拟切片）。确定和选择最好的焦平面，即，具有显着的，重点突出的胰岛β细胞上滚动聚焦旋钮Z轴的数量最多的飞机。一旦选择，StereoInvestigator自动捕获屏幕上显示的图像。随着机动阶段，"虚拟切片"功能顺序捕捉每个光面板内绘制的轮廓（图1A）作为一个鲜明的形象，然后结合所有这些作为一个合并后的图像（图1B）。虚拟切片成像技术使用啶配置即过滤器，广角，让相机捕捉到的所有信号在一定的深度，包括那些不完美中的重点，使最终的虚拟片是一个集成的二维图像，是不是最大从整个胰腺三维重建的投影。平均虚拟切片扫描时间是每个样本30分钟，但可能需要长达一个小时为一个大的胰腺。 </li><li>保存图像后，开始开放ImageJ图像分析。当图像完成加载，并显示在屏幕上，打开和运行的虚拟切片分析的<a href="http://www.jove.com/files/ftp_upload/1970/Supplementary_material 1.pdf">宏（补充材料</a> 1 ）。 </li><li>按照提供的说明一步一步的宏观。消除光散射的"魔棒"和"选择区域"工具创建不受欢迎的文物。不必要的检查，并从图像中减去背景。确定半径最大的胰岛与"行"的工具，使随后的滚珠功能，能有效去除自体荧光和散射光。测试和选择最合适的"低门槛"，捕捉到的小胰岛β细胞群荧光信号（&lt;3X10 4 微米 2） 。测试和选择最合适的"高门槛"，捕捉到的胰岛荧光信号。运行虚拟切片的量化，将测量产生胰岛面积，周长，圆度，和FERET的直径。 </li></ol><img src="/files/ftp_upload/1970/1970Fig1A.jpg" alt="图1a" /> 图1A虚拟切片图像分析的β细胞的整个分布在一个完整的成人胰腺 在10周的一个雄性小鼠胰腺中的单个光学面板的图像下一个2倍的目标。这些图像包括整个小岛的分布，甚至小群的β细胞（&lt;10个细胞）。 <img src="/files/ftp_upload/1970/1970Fig1B.jpg" alt="图1b" /> 图1B虚拟切片图像分析的β细胞的整个分布在一个完整的成人胰腺 一个统一的虚拟切片与右侧背胰和腹胰左侧。比例尺为5毫米。 代表性的成果精通虚拟切片成像胰腺准备将允许所有的有效量化GFP标记作为一个具有明确和独特的小岛的虚拟切片图像的整个胰腺的β细胞。成功的虚拟切片原位全胰腺成像提供的手段，研究和量化的β-细胞，不仅是个人和总胰岛面积，而且胰岛大小分布，区域分析，和增长方式转变以及的。虽然ImageJ维护的能力，同时在虚拟切片图像分析检测所有的β细胞，它也可以控制产生β细胞，进行量化分析，通过限制某些胰岛的尺寸（面积或直径），或由位置。一个标准的分析不包括文物，例如，比一个单一的β-细胞（&lt;170微米）和记录的面积，周长（周边地区的距离），圆（一个圆度的程度，其中1.0代表一个完美的圆）小碎片。 FERET每个胰岛的直径（在一个区域内的最长的距离）检测。虚拟切片分析能力，提高用人的分水岭，区分和分离重叠成单个胰岛的胰岛细胞，根据它们的形状虚拟切片图像。进一步的分析产生了一系列详细的图像，这包括口罩的图像在低和高的强度（图1C），所有推定胰岛大纲，这是每个独立编号及相应的信息表中列出（图1D），和原来的虚拟切片图像（图1B）。选择可以倾斜的小岛，包括无偿光周围的大型结构的量化，或减少总数的胰岛细胞，尤其是小颗粒的微弱信号的阈值时，需要注意的是潜在的技术偏见。 <img src="/files/ftp_upload/1970/1970Fig1C.jpg" alt="图1C" /> 图1C虚拟切片图像分析的β细胞的整个分布在一个完整的成人胰腺 在虚拟切片的荧光颗粒的面具。颜色代码:[1]蓝色:低强度的荧光; [2]绿​​色:中等强度的荧光; [3]红色:高强度的荧光。 <img src="/files/ftp_upload/1970/1970Fig1D.jpg" alt="图1d" /> 图1D的虚拟切片图像，在一个完整的成人胰腺的β细胞的整个分布分析 量化的个别小岛/β-细胞群。请注意，每个胰岛（包括β细胞的小群）是其在相应的图表详细信息，编号。每个结构的四个参数:（1）区（2）周边;（3）循环:这里的人数1.0代表一个完美的圆某种程度的圆度;和（4）FERET的直径:在一个区域内最长的距离。请注意，＃706显示面积只有少数β-细胞分析的决议。分水岭分割检测相邻的小岛组，如所示，并适当的区分不同的胰岛细胞（胰岛1554，1555和1556），他们。 <img src="/files/ftp_upload/1970/1970Fig1E.jpg" alt="图1e" /> 图1E虚拟切片图像分析的β细胞的整个分布在一个完整的成人胰腺 3维虚拟切片分析的情节，面积，周长，和FERET的直径的轴。每个点代表一个小岛。

<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>

完整的胰腺中的胰岛细胞和β细胞集群的虚拟切片图像分析提供了一个大规模的看法，整个小岛分布。发展，随着时间的推移，总胰岛分布的变化，因此可以研究和比较。在我们的胰岛形成（1）分析表明了这样一个例子。在不同时间点（P1 - P21）的综合分析表明新生小鼠的胰腺，胰岛裂变形成。虽然β-细胞的增殖与小鼠的对数正态分布的概率密度函数从P1 - P10适合从P12开始，胰岛分布向左偏离距离...

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<td>Forceps</td>
<td>&nbsp;</td>
<td>Miltex</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
<td>14001-13</td>
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<td>Scissors</td>
<td>&nbsp;</td>
<td>F.S.T</td>
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<td>02s5	</td>
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<td>Large glass slide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>71862-01</td>
<td>75x51x1.2mm</td>
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<td>Large cover glass</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc</td>
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in situ quantification of pancreatic beta-cell mass in mice

跟踪在健康和疾病的特定细胞群的变化是生物医学研究的一个重要目标。监测胰腺β细胞增殖和胰岛生长的过程中是特别具有挑战性。我们已经开发出一种方法来捕获完整的胰腺β细胞的分布与荧光标记的β细胞ImageJ（/ rsb.info.nih.gov / IJ）编写的宏转基因小鼠。胰腺解剖和组织清算，作为一个虚拟切片，捕获整个胰腺后，GFP标记的β细胞的研究。分析包括定量β细胞总面积，胰岛数量和大小分布参考具体参数和每个胰岛β细胞小群的位置。可以绘制整个小岛分布在三个层面，从每个胰岛的大小和形状的分布信息，允许一个整体β细胞面积的变化一目了然的定量和定性的比较。

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

以下协议概述了虚拟切片成像过程中，胰腺癌的夹层和随后所有GFP标记在整个胰腺β细胞的定量。

在小鼠胰腺β-细胞团的原位定量

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 次观看.  University of Chicago. 以下协议概述了虚拟切片成像过程中，胰腺癌的夹层和随后所有GFP标记在整个胰腺β细胞的定量。

视频: 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.

补肾胶囊对糖尿病小鼠的胰岛移植

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 ...

科研

生物学

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.

电离辐射的反应定量的γH2AX灶

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.

计算机辅助大型胰岛质量，粒度分布和结构的可视化和量化

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.

加上质谱肽免疫亲和富集的蛋白质定量

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.

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

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.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（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.

选择反应监测质谱法进行绝对定量蛋白质

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.

结晶纤维素积累高分辨率的量化<em&gt;拟南芥</em&gt;根监视特定组织细胞壁修改

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).

观察与端粒的定量和重复序列用荧光<em&gt;原位</em&gt;杂交（FISH）与PNA探针<em&gt;秀丽隐杆线虫</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.