"在真核细胞中，利用生物信息学和分子生物学方法的建模细菌蛋白质异戊烯"玛丽·路易丝·安德鲁斯奖的癌症研究计划，弗吉尼亚州科学院主办，"血浆中的RNA编辑的生理意义的项目过程中，DIY低温检索设备已测试树突状细胞"由托马斯·Jeffress和凯特·米勒Jeffress纪念信托和国家合同16.740.11.0364（科教部，俄罗斯）。我们想感谢援助与RNA的完整性实验贝丝EOM。

1。大会Cryotolerant设备Cryoboxes检索<ol><li>用钳子，理顺3片面强领带（ 图1A），使L形图1b所示的强领带注:侧。强领带的尺寸可以选择取决于杜瓦颈部直径。 </li><li>确保两个这样的强强烈的领带ţ表带使用皇冠螺栓螺母，垫圈和螺钉形成Cryoscoop基地，在图3（AB）所示（ 图2）注:两强关系是用来允许检索5年半英寸x 5½英寸cryoboxes。 </li><li>使用开槽板（ 图4），强ţ领带皮带上的插槽与插槽对齐开槽板的一端，并确保与帮助希尔曼螺母和螺栓（ 图5），两项合计注:如果开槽板和强烈的领带并没有合作我与预钻孔，您将不得不使用硬质合金钻头孔钻。 </li><li>弯曲的钳子的帮助下，开槽板的另一端， 如图6所示。 （这一步是可选的）。 </li><li>包装用泡沫手柄，更好地掌握设备时，在液氮中， 如图6所示。 （可选） 注意:以高压灭菌前取出泡沫。 </li></ol> 2。大会Cryotolerant设备冷冻管检索 Cryotolerant DIY的检索设备，也可以适应冷冻管检索。这涉及到代不锈钢滤网平底舀出液氮瓶（ 图7）改变。 <ol><li>拧官方螺栓和螺母，开槽板强领带ţ表带附加。 </li><li>开槽板前附加不锈钢滤网，轻轻弯曲的颈部;这将会使设备更容易操纵，如果过滤网唇，用钳子弯唇:用钳子帮助定位在一个角度基地类似于勺子（ 图8）注意过滤。 </li><li>随着开槽板的孔对准过滤器手柄上的洞，并确保滤网帮助希尔曼螺母和螺栓， 如图8所示的注意的:如果过滤不预钻孔，您将需要安排有硬质合金钻头钻孔。 </li></ol> 3。检索的杜瓦Cryoboxes和冷冻管<ol><li>开始，放在低温手套，并删除1个或2个冰柜从杜瓦架到允许的回旋余地。 </li><li>轻轻刮去沿杜瓦底部的直立Cryotolerant DIY检索装有强领带ţ表带基地（ 图5）设备和收集箱fallen个底部的杜瓦罐注:液氮沸腾。立即用温暖的对象接触，包绕在绝缘氮气体（Leidenfrost效果）的对象。冷却装置之前，检索样本，慢慢地浸入到杜瓦设备，并允许液氮冷却，然后再进行上述步骤的设备足够。 </li><li>直立拉设备，沿墙壁杜瓦罐舀出冷冻箱。 </li><li>抓斗从杜瓦底部检索框。 </li><li>检索冷冻管，轻轻刮去杜瓦的底部装有一个过滤器（ 图9）cryoscoop。 </li><li>类似cryobox检索，正直和抢拉设备在过滤器收集的小瓶。 </li></ol> 4。代表结果在基因表达研究中的一个主要挑战是，RNA的质量取决于储存条件冷冻组织标本，提取RNA。 图10显示了样品处理的总RNA的完整性的影响。来自同一组织源（人体脂肪）的总RNA受到不同的样品处理。实验之前，完整的总RNA的preparate显示清晰的28S和18S rRNA基因与28S rRNA的乐队乐队约两倍激烈的18S rRNA带（ 图10，泳道1）。这个比例为2:1（28S:18S）是一个迹象表明，RNA是完整的，而部分降解的RNA出现一抹黑，或者其28S 18S强度比从2:1 5出发。泳道3-4描绘了显着降解的RNA样品。这些RNA样品经反复局部defreezing从杜瓦（方法A）的短期检索，从而建模与长期贮存定期滗由于堕落瓶和盒检索的必要性（ 图10巷3-4 ）。然而，样本存储在杜瓦服务使用自助设备（方法B），因此，没有偏defreezing，包含完整的RNA（ 图10巷2号）。 <img alt="图1" src="/files/ftp_upload/3903/3903fig1.jpg" /> 图1。 A）该基地由两个3双面强领带的尺寸。每一个强烈的领带，包含一个大型预钻孔（实心箭头）和多个较小的孔，每个面（小箭头）。这提供了一个选项，以允许用螺母和螺栓紧固。还小的孔允许在样本检索液氮快速排水B）强烈的领带一边用钳子的帮助，使L形强领带拉直。 <img alt="图2" src="/files/ftp_upload/3903/3903fig2.jpg" /> 图2。强领带ţ表带的用于保护强联系在一起。像强领带，T字表带一体的大型预钻孔（实心箭头）和两个较小的孔（SMaL公司升箭头）上的每一个角落。这可以确保沿长度的T强关系 - 表带使用的螺母和螺栓。这也使T型带固定槽板。 <img alt="图3" src="/files/ftp_upload/3903/3903fig3.jpg" /> 图3。一）每个强领带系T型带的一个角落，沿其长度冠螺栓螺母，螺栓和垫圈。 乙）两强关系获得彼此相邻，以帮助检索5½英寸×5½英寸cryoboxes。小cryoboxes如2英寸x 2英寸，单强领带可以直接固定开槽板，没有一个T-表带。 <img alt="图4" src="/files/ftp_upload/3903/3903fig4.jpg" /> 图4。开槽平板作为手柄使用。开槽板的长度取决于杜瓦的深度。在这种情况下，使用了4英尺开槽板。开槽板的厚度是非常重要的小号自从开槽薄钢板（&lt;14计），使设备薄弱，同时检索充满杜瓦液氮样品。 <img alt="图5" src="/files/ftp_upload/3903/3903fig5.jpg" /> 图5。强关系和T带平底组成，然后开槽板的一端固定在。其余的T表带角落较大的孔对齐平板插槽，并用六角螺栓和螺母紧固。该装置可用于检索从杜瓦cryoboxes。 <img alt="图6" src="/files/ftp_upload/3903/3903fig6.jpg" /> 图6。开槽平板的一端用钳子弯曲，并用泡沫覆盖，以提供更好的抓地力，特别是深杜瓦瓶，在这种情况下所示。 <img alt="图7" src="/files/ftp_upload/3903/3903fig7.jpg" /> 图7。滤网，丝网和不锈钢拉手用于检索从杜瓦冷冻管。如果手柄没有提供一个插槽，钻硬质合金钻头钻一个洞，将需要。 <img alt="图8" src="/files/ftp_upload/3903/3903fig8.jpg" /> 图8。为了便于检索的冷冻管，过滤器一起用钳子杓形滤网的颈部弯曲。如果唇在这种情况下，提供了过滤器，钳子可以用来弯曲。这可确保滤网内没有得到杜瓦捕获。 <img alt="图9" src="/files/ftp_upload/3903/3903fig9.jpg" /> 图9。过滤器的一端固定在平板开槽六角螺母和螺栓。 <img alt="图10" src="/files/ftp_upload/3903/3903fig10.jpg" /> 图10。样品的总RNA的完整性受到不同的样品处理的条件。探讨如何处理C样品的影响RNA的质量onditions，一套来自同一来源的RNA样品受到两种方法的装卸。在使用自助设备的样品检索杜瓦，因此，没有偏defreezing包括RNA的存储方法A。方法B遭受杜瓦短期检索重复的部分defreezing存储在杜瓦的RNA，从而建模与长期存储，检索下跌样品定期调迁。总RNA的完整性，然后用凝胶电泳分析。泳道1:样品前存储杜瓦;巷2:从的杜瓦样品遭受的检索方法A; 3巷，4:样品从遭受的检索方法B杜瓦，M:100 bp的阶梯。

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	<li>Kittel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Cryogenic Engineering. , 41 (1996).</li><li>Edeskuty, F. J., Walter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Stewart Safety in the handling of cryogenic fluids. , (1996).</li><li>Council, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards. , (2011).</li><li>Muyal, J., Muyal, V., Kaistha, B., Seifart, C., Fehrenbach, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+RNA+extraction+techniques+from+frozen+and+fresh+lung+tissues:+checkpoint+towards+gene+expression+studies.">Systematic comparison of RNA extraction techniques from frozen and fresh lung tissues: checkpoint towards gene expression studies.</a> Diagnostic Pathology. 4, 9 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory manual. , (2001).</li><li>Holland, N. T., Smith, M. T., Eskenazi, B., Bastaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+sample+collection+and+processing+for+molecular+epidemiological+studies.">Biological sample collection and processing for molecular epidemiological studies.</a> Mutation Research/Reviews in Mutation Research. 543, 217-234 (2003).</li><li>Lynch, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CELL,+TISSUE+AND+ORGAN+CULTURE+|+Storage+and+Cryopreservation.">CELL, TISSUE AND ORGAN CULTURE | Storage and Cryopreservation.</a> Encyclopedia of Rose Science. , 106-111 (2003).</li><li>Crawford, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plastics Engineering. , (1998).</li><li>Gorenc, B., Tinyou, R., Syam, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Steel designers' handbook. , (1996).</li><li>Harper, C. A., Petrie, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastics+materials+and+processes:+a+concise+encyclopedia.">Plastics materials and processes: a concise encyclopedia.</a> Wiley-Interscience. , (2003).</li></ol>

没有利益冲突的声明。

检索cryoboxes和冷冻管从杜瓦罐底部的最常用的方法之一是迁置到一个备用的容器中的液态氮和拉出样品留在烧瓶或漂浮在倒出的液氮。然而，这是一个不安全的做法，可能会导致低温烫伤或窒息，由于长时间暴露在液氮蒸汽2。其他常见的方式检索堕落的冷冻管涉及使用离心管钳3。然而，黑暗的空间内充满液氮罐的能见度有限，限制了其可操作性。 如上所述?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>希尔曼集团镀开槽平1-3/8×4英尺14计</td><td>当地的五金商店</td><td> N / A </td><td> 1个单位的要求</td></tr><tr><td>皇冠螺栓不锈钢＃6 - 32×3/8英寸扁圆头十字径机螺杆</td><td>当地的五金商店</td><td>型号＃42281 </td><td>需要4个单位</td></tr><tr><td>冠螺栓3/8英寸x 1-1/2英寸镀锌钢护舷垫圈</td><td>当地的五金商店</td><td>型号＃20122 </td><td>需要4个单位</td></tr><tr><td>冠螺栓锌镀＃8-32机螺杆螺帽</td><td>当地的五金商店</td><td>型号＃18512 </td><td>需要4个单位</td></tr><tr><td>辛普森强铁BC40Z 4英寸半基地In.x4 </td><td>本地硬件购物</td><td>型号＃BC40Z </td><td>需要2个单位</td></tr><tr><td>辛普森强铁6英寸x 6英寸ţ表带</td><td>当地的五金商店</td><td>型号:66T </td><td> 1个单位的要求</td></tr><tr><td>奥奈达6"滤网</td><td>本地杂货店</td><td>型号:7859W </td><td> 1个单位的要求</td></tr><tr><td>该的希尔曼集团镀锌六角螺母3/8-16 </td><td>当地的五金商店</td><td>型号＃:810509 </td><td>需要6个单位</td></tr><tr><td>该的希尔曼集团镀锌六角螺栓3/8- 16 </td><td>当地的五金商店</td><td></td><td>需要4个单位</td></tr><tr><td>绝缘泡沫</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr><tr><td>螺杆驱动程序</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr><tr><td>钳子</td><td>当地的五金商店</td><td></td><td> 1个单位的要求</td></tr></tbody></table>

do-it-yourself device for recovery of cryopreserved samples accidentally dropped into cryogenic storage tanks

液氮是无色，无嗅，极冷（-196℃）的液体在压力下保持。它通常被用来作为一个长期的生物材料，如血液，细胞和组织1,2长期储存的低温液体。液氮低温性质，而理想的样品保存，可引起接触活组织快速冷冻-被称为"低温烧伤"2，这可能会导致密切合作，参与从杜瓦瓶样品的存储和检索的人严重冻伤。此外，液氮蒸发，降低空气中的氧气浓度，并可能导致窒息，特别是在密闭空间2。 在实验室，生物样品往往存储在冷冻管或不锈钢架堆放在杜瓦罐1 cryoboxes。提供这些存储架，以防止打滑从机架盒与长轴成底部杜瓦在常规处理。然而，过于频繁，箱或珍贵样品小瓶溜出去和充满液氮罐的底部下沉。在这种情况下，样本可以不厌其烦地检索后液氮转移到一个备用的容器中或丢弃它。然后可以相对安全地从回收空杜瓦盒和瓶。然而，低温液态氮，其扩张速度的性质，使沉船样本检索的危险。它通常被推荐样本检索从未由一个人进行了安全处。另一种方法是使用市售的酷抓斗或钳拉出瓶3。在黑暗的充满液体的杜瓦瓶的能见度有限，但在其使用带来的一个主要的限制。 在这篇文章中，我们描述了一个Cryotolerant DIY的检索设备，这使得从杜瓦样本检索含有低温液体既安全建设第二容易。

Watch this Scientific Journal Video about Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks at JoVE.com

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

在这里，我们提出了成本低，耐用cryotolerant设备样品从充满液氮的杜瓦罐检索。易于施工和设备的模块化设计使得低温储罐的样本检索过程的安全和方便。

做自己动手恢复冻存样品的设备不慎掉落到低温储罐

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

do-it-yourself-device-for-recovery-cryopreserved-samples-accidentally

Research

JoVE Journal

Biology

25.2K 次观看.  George Mason University. 在这里，我们提出了成本低，耐用cryotolerant设备样品从充满液氮的杜瓦罐检索。易于施工和设备的模块化设计使得低温储罐的样本检索过程的安全和方便。

视频: Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

条块索玛的神经元和轴突的一个微流体设备制造

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

科研

生物学

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

微流体装置与Groove的模式，为研究细胞行为

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

一个渐变产生的微流体装置，细胞生物学

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

准备在微流体装置E18条块皮质大鼠神经元

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

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 ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品和环境样品中沙门氏菌的准总体基因体分析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive behaviors and the acquisition of gametes and embryos for physiological or molecular research. Amphibians contribute to one-quarter to one-third of vertebrate research, and of interest in this context is their contribution to the scientific community&#39;s knowledge of reproductive processes and embryological development. However, most of this knowledge is derived from a small number of species. In recent times, the decimation of amphibians across the globe has required increasing intervention by conservationists. The captive recovery and assurance colonies that continue to emerge in response to the extinction risk make existing research and clinical applications invaluable to the survival and reproduction of amphibians held under human care. The success of any captive population is founded on its health and reproduction and the ability to develop viable offspring that carry forward the most diverse genetic representation of their species. For researchers and veterinarians, the ability to monitor and control ovarian development and health is, therefore, imperative. The focus of this article is to highlight the different assisted reproductive techniques that can be used to monitor and, where appropriate or necessary, control ovarian function in amphibians. Ideally, any reproductive and health issues should be reduced through proper captive husbandry, but, as with any animal, issues of health and reproductive pathologies are inevitable. Non-invasive techniques include behavioral assessments, visual inspection and palpation and morphometric measurements for the calculation of body condition indices and ultrasound. Invasive techniques include hormonal injections, blood sampling, and surgery. Ovarian control can be exercised in a number of ways depending on the application required and species of interest.

The study of amphibian biology provides valuable information on the reproductive, physiological, embryological and developmental processes that drive organisms of many taxonomic groups. Here, we present a comprehensive guide on different methodologies that can be used to study ovarian control and monitoring in amphibians.

两栖动物卵巢监测和控制的生殖技术

Ovarian control and monitoring in amphibians require a multi-faceted approach. There are several applications that can successfully induce reproductive ...

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

低温样品加载到魔法角度旋转核磁共振光谱仪中，可保持细胞生存能力

Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. ...

Strategies for Optimization of Cryogenic Electron Tomography Data Acquisition

Cryogenic electron tomography (cryoET) is a powerful method to study the 3D structure of biological samples in a close-to-native state. Current state-of-the-art cryoET combined with subtomogram averaging analysis enables the high-resolution structural determination of macromolecular complexes that are present in multiple copies within tomographic reconstructions. Tomographic experiments usually require a vast amount of tilt series to be acquired by means of high-end transmission electron microscopes with important operational running-costs. Although the throughput and reliability of automated data acquisition routines have constantly improved over the recent years, the process of selecting regions of interest at which a tilt series will be acquired cannot be easily automated and it still relies on the user's manual input. Therefore, the set-up of a large-scale data collection session is a time-consuming procedure that can considerably reduce the remaining microscope time available for tilt series acquisition. Here, the protocol describes the recently developed implementations based on the SerialEM package and the PyEM software that significantly improve the time-efficiency of grid screening and large-scale tilt series data collection. The presented protocol illustrates how to use SerialEM scripting functionalities to fully automate grid mapping, grid square mapping, and tilt series acquisition. Furthermore, the protocol describes how to use PyEM to select additional acquisition targets in off-line mode after automated data collection is initiated. To illustrate this protocol, its application in the context of high-end data collection of Sars-Cov-2 tilt series is described. The presented pipeline is particularly suited to maximizing the time-efficiency of tomography experiments that require a careful selection of acquisition targets and at the same time a large amount of tilt series to be collected.

The increasing demand for large-scale data collection in cryogenic electron tomography requires high-throughput image acquisition routines. Described here is a protocol that implements the recent developments of advanced acquisition strategies aimed at maximizing the time-efficiency and throughput of tomographic data collection.