这项研究是由赠款支持，援助青年科学家（一）（＃16680015），为探索性研究赠款，援助（＃16653065），于优先领域的科研赠款援助（＃18023022＃ 18016012和18053015＃）由教育部，文化，体育，科学和技术在日本，格兰特推广健康科学的基础研究，生物医学创新研究所（NIBIO），格兰特的援助赈灾基金拨款援助，从日本神经信息学中心（NIJC），理化学研究所

<ol><li>光/暗转换测试使用的仪器，包括划分成大小相等的两个部分，一个门分区（大原公司，东京）一笼（21x42x25厘米）。 </li><li>小鼠被安置在一个房间里，每笼12小时随意获取食物和水的光/暗周期（上午7:00灯）三个四个。理想的情况下，两个控件和两个突变都住在一起，通过重新组织，尽快为他们在断奶时基因型的小鼠。行为测试之间进行的上午9:00和下午6:00所有的笼子转移到小鼠的行为测试室的第一次审判开始前30分钟。 </li><li>一室明亮的白色二极管照明（390勒克斯），而其他商会是黑暗（2勒克斯）。小鼠被放置到阴暗的一面，门被打开后，鼠标是由红外摄像机检测到3秒自动。门使用，因此，老鼠不进入光室后，立即释放他们的动机摆脱实验者，由于延迟进入光室可作为焦虑样行为的指标。 </li><li>小鼠允许两院之间的门打开10分钟的自由移动。用于采集和分析的行为数据（图像LD4）的应用程序是基于对公共领域的图像J计划（在美国国立精神卫生研究所byWayne Rasband http://rsb.info.nih.gov/ij /），这是刚宫川（通过奥哈拉公司，日本东京）的修改。 </li><li>距离在每个分庭，总数的过渡，在每个分庭花的时间，延迟进入光chamberare记录图像LD4方案。 </li><li>每次试验后，所有分庭具有超强的次氯酸水清洗，以防止对嗅觉线索为基础的偏见。 </li></ol>

<ol>
	<li>Austin, C. P., Aiba, A., Takao, K., Miyakawa, T., Takao, K., Yamasaki, N., Miyakawa, T., Crawley, J. N., Crawley, J. N., Crawley, J., Goodwin, F. K., Miyakawa, T., Crabbe, J. C., Wahlsten, D., Dudek, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36, 921-924 (2004).</li></ol>

所有的程序获得批准使用动物保健委员会和日本京都大学的。宫川是刚与鸟，日本科学技术振兴机构，日本埼玉县。

虽然所有的小鼠基因组序列已经确定，大部分这些基因的功能尚不清楚。基因定位技术，但是，可以用来删除或操纵在小鼠的特定基因（AIBA等，2007; Austin等人，2004年）。对某一具体行为的特定基因的影响，然后可以由突变小鼠的行为进行分析（隆夫和宫川，2006年;。隆夫等，2007）。作为行为的突变小鼠的表型试验，光/暗的过渡测试是使用最广泛的测试，以测量在小鼠焦虑行为（克劳利，2000年）之一。该测试是基于对小鼠自然厌恶明亮的照明领域，并以新颖的环境自发的探索行为（克劳利，1985）。测试是敏感的抗焦虑药物治疗（克劳利，1985年）。该装置由一个黑暗室和一个明亮的照明室。一个受限制的开放，高3厘米，宽5厘米，连接两院。小鼠允许两院之间自由移动。到明亮的商会参赛作品的数量和持续时间花在有明亮的空间在小鼠的焦虑指数。 克劳利和他的同事们（克劳利和Goodwin，1980年）最初是由光/暗转换测试。其原始版本和我们的测试之间的差异有两个。首先，光腔比原版本中的暗腔较大，而两院的大小是相同的，在我们的测试版本的。其次，在原来的版本，光室不设上限和光室的墙壁都是透明的（克劳利和Goodwin，1980），而我们使用光室的墙壁和天花板的白色不透明塑料。这些差异，即光腔的大小和开放性，允许同时检测的明亮空间的焦虑，以及开放空间的原始版本在测试焦虑。然而，在我们的实验室，开放空间的焦虑般的行为在小鼠是在高架十字迷宫测试。不透明的墙壁和天花板，光腔的大小，和我们的具体的光/暗转换测试协议是专门检测原来的版本相比，明亮的空间焦虑。 虽然光/暗转换测试和高架十字迷宫都用于评估类似焦虑的行为，其结果并不总是一致的。例如，特定的脑钙调神经磷酸酶基因敲除小鼠光在光/暗转换测试室花费的时间减少量，但增加的时间在高架十字迷宫张开双臂量（宫川等，2003） 。我们假设我们的光/暗转换测试和高架十字迷宫试验评估焦虑样行为的不同方面，如明亮的空间焦虑和开放空间的焦虑样行为，。为了检验这一假设，我们用一组在这两项测试中使用相同的小鼠，收集大量的数据。在我们的实验室，我们进行行为测试电池，涵盖诸多领域，行为意义的基因屏幕。测试电池需要几个星期才能完成。在测试期间，鼠标笼存放在我们的设备机架上。如果我们从其他机构的实验室老鼠是运到，他们习惯于我们的设施环境，至少1周。习惯期后，小鼠的一般健康检查和筛选神经系统（宫川等人，2003年），和这些简单的考试后，小鼠受到光/暗转换测试。在一个健全的证明后，习惯性试验室的30分钟的试验室进行测试，如在电影中所示。 我们已评估超过60株突变小鼠基因工程使用在电影中的协议，并有3000多个小鼠（包括野生型和突变型小鼠）的原始数据的大集。我们进行了因素分析，采取了大量的原始数据集的优势。事实上，我们发现不同的因素，从每个测试（未发表数据），指数（未发表数据）之间虽然有一些显着的相关性..作为一个公共数据库，我们现在建设的一部分，这些数据将被披露。 在我们的测试电池中，野生型窝通常用来作为对照。作为背景应变，常用C57BL/6J小鼠。我们用来测试电池控制的突变小鼠C57BL/6J小鼠中收集的数据。从C57BL/6J小鼠在光/暗转换测试得到的指数如下（N = 795;平均教育统筹局局长）;距离：846.5 8.88厘米，在光室，在黑暗中腔1478.5 9.53厘米;持续时间花：215.8 1.85 395.8 1.86 S的光，在黑暗中的S;车厢之间的转换数量：30.52 0.43;延迟进入光室：64.94 2.40秒各地的分庭入口处小鼠的运动检测相机，并在C所花费的时间计算hambers。正因为如此，在光度过的时间平均持续时间的总和，在黑暗的车厢大于整个工期的测试（600）。 在我们的协议中，鼠标放置在暗室内。三秒钟后，鼠标放置在黑暗中商会，商会之间的门自动打开，鼠标可以自由移动两院之间。 C57BL/6J小鼠花费更少的时间比在黑暗中商会（P &lt;0.0001，持续时间，如果时间花在轻光腔：215.8 1.85小号，在黑暗中度过的时间期限：316.3 2.11小号的时间花费在黑暗中不不包括初始时间花费在黑暗中前首次进入光室]，N = 795，配对t检验）。这表明C57BL/6J小鼠倾向于避免光室，光花的时间是一个良好的焦虑样行为的指标。 从不同菌株的突变小鼠中取得的成果很容易比较，因为所有的数据都使用相同的协议。要获得一个可以很容易地复制和与其他突变体的数据相比，突变小鼠的菌株分型结果，行为测试方法应如实验室之间可能完全相同。 Crabbe和他的同事测试鼠标行为和实验室环境中使用完全相同的近交系和一个空的突变株之间的相互作用。在他们的研究，严格规范的仪器，测试协议，许多环境变量。尽管标准化，他们发现在整个实验室的行为进行了系统性的差异，虽然他们也发现在所有的行为（克拉布等，1999）的应变差异。他们报告说，不可控变量和特征的突变体的实验可能产生的结果，则是千变万化的，以一个特定的实验室。实验室之间存在的程序上的差异使得它难以复制或实验室之间的比较结果。在这里，我们目前的光/暗转换测试作为一部电影，使该协议的细节可以证明我们的协议。 建立视觉文档的协议将有助于更好地理解实验程序的细节，允许在实验室和不同菌株的突变小鼠的行为表型的比较使用的协议的标准化评估使用这个测试。电影的其他协议，如高架十字迷宫，开放式的现场测试，porsolt强迫游泳测试，怕空调，我们在我们的行为测试电池的使用，目前正在作为未来视频杂志文章的出版。

light/dark transition test for mice

虽然所有的小鼠基因组序列已经确定，我们不知道大多数这些基因的功能。基因定位技术，然而，可用于删除或操纵在小鼠体内的特定基因。突变小鼠的行为进行分析，然后确定特定基因上的一个特定的行为的影响。作为行为的突变小鼠的表型试验，光/暗的过渡测试是使用最广泛的测试，以衡量状的焦虑的小鼠行为之一。该测试是基于对小鼠自然厌恶明亮的照明领域，并以新颖的环境中自发探索行为。测试是敏感的抗焦虑药物治疗。该装置由一个黑暗室和一个明亮的照明室。小鼠允许两院之间自由移动。到明亮的商会参赛作品的数量和持续时间花在有明亮的空间在小鼠的焦虑指数。要获得一个可以很容易地复制和与其他突变体的突变小鼠的菌株分型结果，行为测试方法应如实验室之间可能完全相同。然而，程序实验室之间存在的差异使其难以复制或实验室之间的比较结果。在这里，我们目前的光/暗转换测试作为一部电影，使该协议的细节可以证明我们的协议。在我们的实验室，我们已经评估了超过60株的使用在电影中的协议的突变小鼠。作为一个公共数据库，我们现在建设的一部分，这些数据将被披露。 形象化的协议将有助于了解整个实验过程的细节，允许各实验室和不同菌株的突变小鼠的行为表型的比较中使用的协议的标准化评估使用这个测试。

Watch this Scientific Journal Video about Light/dark Transition Test for Mice at JoVE.com

Light/dark Transition Test for Mice

光/暗转换的测试是使用最广泛的测试，以衡量状的焦虑的小鼠行为之一。在这里，我们提出了一个电影，显示了我们如何进行测试的详细程序。

光/暗转换测试小鼠

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Research

JoVE Journal

Biology

51.7K 次观看.  Graduate School of Medicine, Kyoto University. 光/暗转换的测试是使用最广泛的测试，以衡量状的焦虑的小鼠行为之一。在这里，我们提出了一个电影，显示了我们如何进行测试的详细程序。

视频: Light/dark Transition Test for Mice

Elevated Plus Maze for Mice

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given gene on a specific behavior can be determined by behavioral analysis of mutant mice. If a target gene is expressed in the brain, behavioral phenotype of the mutant mice could elucidate the genetic mechanism of normal behaviors. The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior. The test is based on the natural aversion of mice for open and elevated areas, as well as on their natural spontaneous exploratory behavior in novel environments. The apparatus consists of open arms and closed arms, crossed in the middle perpendicularly to each other, and a center area. Mice are given access to all of the arms and are allowed to move freely between them. The number of entries into the open arms and the time spent in the open arms are used as indices of open space-induced anxiety in mice. Unfortunately, the procedural differences that exist between laboratories make it difficult to duplicate and compare results among laboratories. Here, we present a detailed movie demonstrating our protocol for the elevated plus maze test. In our laboratory, we have assessed more than 90 strains of mutant mice using the protocol shown in the movie. These data will be disclosed as a part of a public database that we are now constructing.
Visualization of the protocol will promote better understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used in different laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test. 


The elevated plus maze test is one of the most widely used tests for measuring anxiety-like behavior in mice. Here, we present a movie showing the detailed procedures for conducting the test.

对小鼠高架十字迷宫

Although the mouse genome is now completely sequenced, the functions of most of the genes expressed in the brain are not known. The influence of a given ...

科研

生物学

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been understood at molecular level. One of the mechanisms mediating the effect of DNA variants on phenotypes is gene expression, which has been shown to be particularly relevant for complex traits1.
This method tests in a cellular context the effect of specific DNA sequences on gene expression. The principle is to measure the relative abundance of transcripts arising from the two alleles of a gene, analysing cells which carry one copy of the DNA sequences associated with disease (the risk variants)2,3. Therefore, the cells used for this method should meet two fundamental genotypic requirements: they have to be heterozygous both for DNA risk variants and for DNA markers, typically coding polymorphisms, which can distinguish transcripts based on their chromosomal origin (Figure 1). DNA risk variants and DNA markers do not need to have the same allele frequency but the phase (haplotypic) relationship of the genetic markers needs to be understood. It is also important to choose cell types which express the gene of interest. This protocol refers specifically to the procedure adopted to extract nucleic acids from fibroblasts but the method is equally applicable to other cells types including primary cells.
DNA and RNA are extracted from the selected cell lines and cDNA is generated. DNA and cDNA are analysed with a primer extension assay, designed to target the coding DNA markers4. The primer extension assay is carried out using the MassARRAY (Sequenom)5 platform according to the manufacturer's specifications. Primer extension products are then analysed by matrix-assisted laser desorption/ionization time of-flight mass spectrometry (MALDI-TOF/MS). Because the selected markers are heterozygous they will generate two peaks on the MS profiles. The area of each peak is proportional to the transcript abundance and can be measured with a function of the MassARRAY Typer software to generate an allelic ratio (allele 1: allele 2) calculation. The allelic ratio obtained for cDNA is normalized using that measured from genomic DNA, where the allelic ratio is expected to be 1:1 to correct for technical artifacts. Markers with a normalised allelic ratio significantly different to 1 indicate that the amount of transcript generated from the two chromosomes in the same cell is different, suggesting that the DNA variants associated with the phenotype have an effect on gene expression. Experimental controls should be used to confirm the results.

Genetic associations often remain unexplained at a functional level. This method aims to assess the effect of phenotype-associated genetic markers on gene expression by analyzing cells heterozygous for transcribed SNPs. The technology allows accurate measurement by MALDI-TOF mass spectrometry to quantify allele-specific primer extension products.

等位基因特异性基因表达检测，测试的遗传协会的功能基础

The number of significant genetic associations with common complex traits is constantly increasing. However, most of these associations have not been ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy techniques. However, as the image is acquired in a very different way than normal, by building up an image molecule-by-molecule, there are some significant challenges for users in trying to optimize their image acquisition. In order to aid this process and gain more insight into how STORM works we present the preparation of 3 test samples and the methodology of acquiring and processing STORM super-resolution images with typical resolutions of between 30-50 nm. By combining the test samples with the use of the freely available rainSTORM processing software it is possible to obtain a great deal of information about image quality and resolution. Using these metrics it is then possible to optimize the imaging procedure from the optics, to sample preparation, dye choice, buffer conditions, and image acquisition settings. We also show examples of some common problems that result in poor image quality, such as lateral drift, where the sample moves during image acquisition and density related problems resulting in the 'mislocalization' phenomenon.

We describe the preparation of three test samples and how they can be used to optimize and assess the performance of STORM microscopes. Using these examples we show how to acquire raw data and then process it to acquire super-resolution images in cells of approximately 30-50 nm resolution.

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

检测选择性剪接的过程中上皮 - 间质转化

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.