The authors would like to acknowledge the following funding sources: NIH R01-AR063702, R21-AR064941, K99-AR067283, and T90-DE021989.

康涅狄格卫生中心的机构动物护理和使用委员会的批准大学所有的动物程序。 1.固定和嵌入<ol><li>通过安乐死的 CO 2窒息或其他认可的方法动物。 </li><li>收获的兴趣，在10％中性缓冲的福尔马林的组织（ 如，肢体，椎骨，等等），并置于4℃直到正确固定。特别注意前固定保持一致的解剖学位置。例如，在一致的屈曲，内旋固定含接头四肢，外旋角11。通常情况下，整个修复四肢鼠标为1 - 3天。 注意:福尔马林是有毒的，应在通风橱而穿着适当的个人防护装备进行处理。 注意:固定的时间范围取决于样品的大小。如果实验允许，切开骨头将提高骨髓车厢固定。有些标本可能需要灌注固定15，以减少背景荧光（骨髓内如微弱的荧光信号）。 </li><li>传递从福尔马林标本到在1×PBS中制成并孵育12 30％蔗糖 - 在4℃下24小时。 注意:一旦温育12 - 24小时，试样然后可在-80℃放置长期储存。被推荐用于在其中研究人员打算在同一个块中嵌入多个样本，但可能没有收获所有这些样品的同时实验存储的这种方法（ 例如 ，用多个时间点实验）。 </li><li>从蔗糖中取出标本和解剖走任何多余的组织。 </li><li>填充cryomold（模具的大小是依赖于试样的尺寸）与冷冻包埋介质方式的一部分。放置在模具样品使得切割平面对感兴趣区域是与模具的底部平行。 注意:特定组织位置和方向的一个例子可以在图2A中找到- B。 </li><li>放置cryomold到一张干冰直到冷冻包埋介质冻结的薄层，并随后固定在试样到位。装满冷冻包埋剂，同时保持对干冰颗粒的cryomold的cryomold的剩余量。 </li><li>放置在含有干冰2-甲基丁烷预冷却的容器cryomold。一旦样品完全冷冻，取出cryomolds并甩掉多余的2-甲基丁烷。包裹在玻璃纸和地点cryomolds在-20℃或-80℃冷冻贮存。 注意事项:储存在低温包埋剂时，特别是在-20°C的样品可以干出随着时间的推移。我们建议的样品存储时间超过1 - 2月的冷冻于-80℃或在-80℃下30％蔗糖包埋介质（参见步骤1.3）。 </li></ol> 2.磁带稳定的组织切片<ol><li>从冰箱中取出样品块和cryosta地方吨，-20和-25°C之间设置温度。 </li><li>从cryomold取出块和修剪多余的冷冻用刀片嵌入网上平台。 </li><li>放置一些冷冻包埋介质到样品光盘，然后对准块，使得样品盘的表面是平行于该块从而建立适当的切割平面对感兴趣区域的底表面上。 </li><li>放置在试样圆盘在低温恒温器，并允许在冷冻包埋介质冻结。 </li><li>放置在试样圆盘到样品头和调整头部平行于试样块的表面上，使得刀片切割。 </li><li>修剪块下降到感兴趣的区域内的一个电平，并从该块的表面刷掉任何屑。 </li><li>切下一块cryotape大到足以覆盖在低温恒温器的兴趣和预寒意区域。 注:多个片可切一致尺寸和cryost内存储在切片中。 </li><li>抓住用钳子然后将磁带上的块粘的一面朝下非粘性银/金片胶带取下cryotape非黏附后盾。 </li><li>施加压力用辊子（ 图2C）的cryotape。 </li><li>做一个部分 - 无论是使用自动马达或恒温器的手动切割轮（5 8微米）。 注:这是很好的做法，以保持cryotape的边缘，因为它涉及到舞台上，这样切片（ 图2D）期间，部分不脱落阶段。 </li><li>放置节组织方上来就低温恒温器内的塑料显微镜载玻片。 </li><li>除去从低温恒温器的塑料滑动并允许冷冻包埋介质融化，使得部分附着在滑动的表面上。 </li><li>重复切片的连续切片或其他感兴趣的地区。 </li><li>一旦完成，存储部分在幻灯片框T 4和-20或-80℃取决于所需的存储的长度和下游的应对措施。例如，使用将要染色的酶活性的幻灯片（见5.2 - 5.3）在一个月内，如果保存在4℃。 </li></ol> 3.部分以显微镜载玻片粘附<ol><li>紫外线固化型粘合剂的方法。 <ol><li>标签的显微镜载玻片。 注意:对于自动化程度的提高，样品和幻灯片可以用条形码标记。 </li><li>应用紫外线活化光学粘合剂的一滴两个载玻片，并使用一个塑料载玻片的边缘横跨载玻片的表面上扩散粘合剂的薄层。 </li><li>切断银/金选项卡，并奠定了录音部分组织朝上的第一张幻灯片的粘合剂。从一个边缘到另一个，以尽量减少带下方截留气泡的形成适用部分中的滚动。 注意:第一滑动用于的t预湿侧他带放置在第二个（永久）幻灯片（步骤3.1.4）之前。 </li><li>从第一滑动取出录音部分并将其放置在第二滑动（ 图3A）的粘合剂层上。再次，注意避免气泡滞留。 注:此步骤需要练习才能掌握。 </li><li>一旦为给定的实验部分的适当数量被放置在每个载玻片，擦去从周围区域的多余的粘合剂。 </li><li>仔细检查每节气泡或纤维胶带下面。执行显微镜或放大镜下此检查。如果有障碍物，取出个别部分，去除障碍物，然后将其重新应用到载玻片。 </li><li>一旦确定有绕带部分下没有障碍物，放置下在UV黑光显微镜载玻片5 - 10分钟以交联粘合剂。 </li></ol></li><li>壳聚糖胶粘剂的方法。 <ol><li>日准备Ë1％的壳聚糖胶粘剂。通过在100ml去离子水中溶解0.25毫升浓乙酸制备乙酸溶液（0.25％V / V）。溶解1g在100毫升乙酸溶液壳聚糖粉末，搅拌该溶液的O / N或直到所有的脱乙酰壳多糖粉末溶解。 注意:溶液是稳定在室温。 </li><li>存为将被放置在滑动每个部分脱乙酰壳多糖溶液的下降。 </li><li>切断带的银/金标签，每个录音部组织的一面朝上放置到粘合剂（ 图3A）。使用非常谨慎，以避免气泡滞留。 </li><li>一旦所有的部分被放置在滑动，倾斜在其长边滑动向上和过度壳聚糖拖动到使用镊子的底部边缘。 </li><li>放置载玻片在此方向上的纸巾的顶部在滑动箱。然后重力会导致过量的壳聚糖向下落到毛巾，得到脱乙酰壳多糖的平坦和均匀的层。 </li><li>解放军CE其盖在冰箱Ø撑开滑动盒/ N，使壳聚糖干燥。 注:请参阅表1两种胶粘剂方法之间的比较。 </li></ol></li></ol>参考标记以幻灯片4.应用<ol><li>通过将绿色50微升和50微升在100微升的水红色微球制备参比标识物的溶液。储存在4℃下在黑暗中的解决方案。 </li><li>将10微升或20微升吸管尖到微球的解决方案。添加微球体溶液到邻近感兴趣的区域（ 图3C）每个部分的一小滴。注意不要让感兴趣的区域内的微球。 </li><li>在干燥的RT幻灯片（避光）30分钟，如果处理当日的幻灯片。如果不是这样，放置在一个滑动箱的幻灯片在4℃。 注意:只要事先微球体溶液干燥到水化滑动时，英里在多轮染色/成像crospheres将继续坚持以幻灯片。 </li></ol> 5.染色的多发注:通过选择成像，染色，并重新映像步骤的兼容序列，可以检测并在同一组织部分共定位许多生物信号。每轮成像/染色/重新映像的具有用于特定组织学问题有待开发。成像/染色/重新映像序列通常涉及获取在第一轮的成像，随后荧光多路复用免疫染色和多轮的酶活性污渍的内源荧光信号（ 例如 ，蜂窝GFP，矿化染料， 体内成像探针）。最后，部分可以用显色染料（ 如 H＆E，甲苯胺蓝，番红O 等 ）要突出组织结构进行染色。改编自列于本节是自定义的方法市售的协议。 <ol><li> 钙黄绿素蓝染色累计矿物 注:钙黄绿素蓝染色产生一个一致的矿物表面，其适合于不像暗场或微分干涉对比（DIC）成像自动图像分析。用钙黄绿素蓝染色的问题是，荧光光谱的四环素和地美环素标记重叠。 因此，如果样品中含有这些矿物标签，图像在第一轮与钙黄绿素蓝染色之前成像的标签。 <ol><li>如果组织内的内源性信号，在此之前，染色一步成像，从以前的成像一轮充满1XPBS一个罐子科普林淹没的幻灯片，直到盖玻片掉下来的幻灯片了。捞出晾干盖玻片。 </li><li>制备30毫克/毫升的钙黄绿素蓝溶液在2％的NaHCO 3（由2克碳酸氢钠溶于100毫升水制成2 O和调整ING与盐酸pH值为7.4）。分装和储存在-20°C的解决方案。 </li><li>浸入含有1x PBS中的科普林缸幻灯片15分钟以水合的部分中，如果尚未从先前轮水合。 </li><li>取出幻灯片和干燥的背面和边缘用纸巾。 </li><li>应用100 - 500微升每滑动钙黄绿素蓝溶液并孵育在室温潮湿箱10分钟。 </li><li>冲洗在1×PBS中的幻灯片10分钟3变化。 </li><li>应用〜200微升50％甘油在1X PBS每张幻灯片并安装盖玻片。 </li><li>去除多余的甘油和干燥的幻灯片的底部。 </li></ol></li><li> 抗酒石酸酸性磷酸酶（TRAP）的酶活性染色 注:TRAP是通过在造血谱系包括破骨细胞多种细胞类型中表达。这里介绍的TRAP染色使用黄色荧光酸性磷酸酶底物。 某些荧光信号将OVerlap与自定义黄色滤镜集，包括四环素/去甲金霉素矿化标签钙黄绿素蓝染色和DAPI染液。 <ol><li>从以前的成像一轮充满1X PBS一个罐子科普林幻灯片淹没，直到盖玻片掉下来的幻灯片了。捞出晾干盖玻片。 </li><li>通过溶解9.2克无水乙酸钠和水11.4克酒石酸二钠二水合物，并使最终体积达1,000ml制备缓冲液1。将pH调节至4.2，用浓冰醋酸（〜1.5 - 2毫升）中。储存在4溶液 °C可保存12个月。 </li><li>通过在水中溶解40毫克亚硝酸钠和使最终体积至1ml制备缓冲区2。储存在4℃下长达1周溶液。 </li><li>关于染色的那一天，通过混合7.5毫升缓冲液1和150微升缓冲液2的制备反应缓冲液。 </li><li>应用的反应缓冲液无基材到滑动S表示10 - 15分钟以在较低的pH平衡的组织。 注意:典型体积是〜200微升，但需要足够大以覆盖所有部分。 </li><li>稀释1:50和1之间的黄色荧光酸磷酸酶底物:在反应缓冲液100。 注:稀释将由样品内TRAP活性决定。 </li><li>倒入反应缓冲断幻灯片，并与黄色荧光底物，以幻灯片应用的反应缓冲液。 </li><li>放置幻灯片下在UV黑光〜5分钟，以激活所述衬底。 注:孵育时间可取决于样品中TRAP活性变化。 </li><li>冲洗在1×PBS中的幻灯片10分钟3变化。 </li><li>应用〜200微升50％甘油在1X PBS每张幻灯片并安装盖玻片。 </li><li>去除多余的甘油和干燥的幻灯片的底部。 注:酸性TRAP缓冲区将脱钙的部分。该decalcifica的好处化的是，某些颜色将可再次下行染色（ 如矿化标签）。例如，钙黄绿素蓝染色将TRAP染色过程中被除去。因此，DAPI染液可以在这个频道后续一轮中成像。 </li></ol></li><li> 碱性磷酸酶（AP）的酶活性染色 注:参与矿化，包括成骨细胞和软骨细胞的矿化表达AP多种细胞类型。在这个协议中使用的耐晒大红衬底引出两个荧光红色和红色的发色信号。正因为如此，显色底物将淬灭在基板沉淀的区域的荧光信号。因此，最好是成像可以由AP底物被淬火的荧光信号后执行此污渍。 <ol><li>从以前的成像一轮充满1X PBS一个罐子科普林幻灯片淹没，直到盖玻片掉下来的幻灯片了。雷莫ve和干燥盖玻片。 </li><li> 12.1克三溶解于100ml水中制备的1M Tris。调节pH值至9.5，用1M的NaOH。 </li><li>通过在100毫升的去离子水20.33克的 MgCl 2溶解制备的1M MgCl 2的六水合物。 </li><li>通过在100ml去离子水中溶解11.68克氯化钠制备2M氯化钠。 </li><li>制备100×（20毫克/毫升）的库存固红TR盐通过在50ml的去离子水1被测定粉末溶解溶液。使在-20℃将100μl等分试样并储存。 </li><li>通过在50ml N，N-二甲基甲酰胺500毫克粉末溶解制备100×（10毫克/毫升）酚AS-MX磷酸盐原液。使在-20℃将100μl等分试样并储存。 </li><li>关于染色的那一天，通过加入1ml的1M Tris，0.5毫升的1M MgCl 2的，0.5毫升2M氯化钠制备AP缓冲液和使终体积，使用去离子水10毫升（最终浓度:100毫三，50毫氯化镁 ，100mM的NaCl）中。 </li><li>将AP buffer每张幻灯片和孵化在湿度室中在室温10分钟。 </li><li>通过稀释固红TR盐和色酚AS-MX的100倍股在AP缓冲液为1x准备AP底物缓冲。 </li><li>倾AP缓冲液断幻灯片和与AP底物缓冲液替换。孵育幻灯片5 - 在室温湿度室10分钟。 注:孵育时间将通过样品的AP活性决定。 </li><li>洗涤滑动3倍于1×PBS中，每次5分钟。 </li><li> （:1000稀释的DAPI在50％甘油在1×PBS中的1）安装盖用DAPI复染溶液滑倒。 </li></ol></li><li> 甲苯胺蓝O（TB）染色显色 注意:由于所有以前的步骤都在50％甘油/ PBS溶液下临时含水安装成象，显色步骤还含水条件下成像。然而，染色溶液可以容易随时间浸出。因此，它被尽快染色后提示到图像的甲苯胺蓝。<ol><li>淹没从先前的成像轮在装满去离子水的科普林缸滑动，直到盖玻片落在从幻灯片了。捞出晾干盖玻片。 </li><li>除去盖玻片后，更换新鲜的水和孵育载玻片至少10分钟，以使从PBS盐充分从组织中移除。 </li><li>制备在去离子水中的0.025％结核病溶液。 </li><li>放置幻灯片1结核病的解决方案 - 5分钟。 注:孵化时间是依赖于用户的偏好，但应保持在所有的样品是一致的。 </li><li>地方滑入科普林缸用离子交换水和洗涤载玻片3倍，每次5分钟。 </li><li>用30％甘油溶于去离子水山盖玻片（非1X PBS作为这将导致污渍从组织浸出）。注:甘油封固剂可以与果糖糖浆，这有助于防止来自组织污渍扩散取代。至制备果葡糖浆，在10毫升的去离子水和热溶解30克果糖至60℃直到溶解。 </li></ol></li></ol> 6.成像的多发<ol><li>加载滑入显微镜托盘（ 图3D）。 注:托盘装有弹簧。 </li><li>插入托盘成滑动扫描显微镜（ 图3E）的托盘堆叠。 </li><li>单击概要文件列表中加载配置文件与适当的暴露时间为每个荧光每张幻灯片。点击幻灯片名手动命名每张幻灯片或有阅读软件提供的幻灯片标签上的条形码。点击开始预览扫描按钮拍摄的幻灯片预览图像。 </li><li>设置感兴趣的区域组织中检测向导并保存以供随后的几轮成像。一旦每张幻灯片是安装程序，点击开始扫描按钮开始扫描过程。 注:系统最多可容纳100 SLI宫一次。 </li><li>一旦成像完成后，从各个通道和成像为圆形或的.tif .jpg文件图像的组装和分析导出图像。 </li></ol> 7.图像大会<ol><li>手动方法使用Photoshop（或类似的图像编辑软件能够产生分层图像）。 <ol><li>从每个成像一轮打开各个通道的图像。 </li><li>组装各个图像为一体的合成图像中的图层。要做到这一点，请单击图层的图层面板中的一个图像。然后拖动层到另一幅图像。这个拖放操作将创建图像中的新图层。这样做对所有其它图像。另外，使用脚本（文件&gt;脚本&gt;加载文件到堆栈...）通过加载多个图像文件中的图像栈层来自动完成这一过程。 </li><li>一旦每个图像在合成图像中列为个别层，在该层的名称双击来重命名拉Y一代需要辨别不同的通道。 </li><li>为了通过每一层看到，并创建一个真正的合成图像，改变混合模式从"正常"到"屏幕"图层面板中的每一层。 注意:要加快这一步，改变单一的层筛选，然后在图层上点击右键，选择"复制图层样式"，然后突出显示所有其它图层，然后选择"粘贴图层样式"的画面风格也适用于其他层。 </li><li>如果需要的话，减少的不透明度（变化值10 - 40％）的发色层（ 即 ，甲苯胺蓝），以更好地通过生色层可视化的荧光信号。 注:本协议所使用的瓷砖扫描显微镜持有3边缘滑动，从而最大限度地减少旋转，可以每一轮成像过程中出现的滑动量。因此，它是非常容易为用户手动对准图像，而不必以旋转衍生的图像从不同的轮次成像。 </li></ol></li></ol>

<ol>
	<li>Schofield, P. N., Vogel, P., Gkoutos, G. V., Sundberg, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+elephant:+histopathology+in+high-throughput+phenotyping+of+mutant+mice.">Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice.</a> Dis Model Mech. 5 (1), 19-25 (2012).</li><li>Adissu, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+reveals+correlative+and+unique+phenotypes+in+a+high-throughput+mouse+phenotyping+screen.">Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen.</a> Disease Models and Mechanisms. 7 (5), 515-524 (2014).</li><li>Johnson, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virtual+histology+of+transgenic+mouse+embryos+for+high-throughput+phenotyping.">Virtual histology of transgenic mouse embryos for high-throughput phenotyping.</a> PLoS Genet. 2 (4), e61 (2006).</li><li>Ayadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+large-scale+phenotyping+initiatives:+overview+of+the+European+Mouse+Disease+Clinic+(EUMODIC)+and+of+the+Wellcome+Trust+Sanger+Institute+Mouse+Genetics+Project.">Mouse large-scale phenotyping initiatives: overview of the European Mouse Disease Clinic (EUMODIC) and of the Wellcome Trust Sanger Institute Mouse Genetics Project.</a> Mamm.Genome. 23 (9-10), 600-610 (2012).</li><li>Utreja, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+matrix+response+of+temporomandibular+cartilage+to+mechanical+loading.">Cell and matrix response of temporomandibular cartilage to mechanical loading.</a> Osteoarthr Cartil. 24 (2), 335-344 (2016).</li><li>Dyment, N. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gdf5+progenitors+give+rise+to+fibrocartilage+cells+that+mineralize+via+hedgehog+signaling+to+form+the+zonal+enthesis.">Gdf5 progenitors give rise to fibrocartilage cells that mineralize via hedgehog signaling to form the zonal enthesis.</a> Dev.Biol. 405 (1), 96-107 (2015).</li><li>Lalley, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+biomechanical+and+biological+outcomes+in+the+MRL/MpJ+murine+strain+following+a+full-length+patellar+tendon+injury.">Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury.</a> J.Orthop.Res. 33 (11), 1693-1703 (2015).</li><li>Breidenbach, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablating+hedgehog+signaling+in+tenocytes+during+development+impairs+biomechanics+and+matrix+organization+of+the+adult+murine+patellar+tendon+enthesis.">Ablating hedgehog signaling in tenocytes during development impairs biomechanics and matrix organization of the adult murine patellar tendon enthesis.</a> J.Orthop.Res. 33 (8), 1142-1151 (2015).</li><li>Ushiku, C., Adams, D., Jiang, X., Wang, L., Rowe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Bone+Fracture+Repair+in+Mice+Harboring+GFP+Reporters+for+Cells+within+the+Osteoblastic+Lineage.">Long Bone Fracture Repair in Mice Harboring GFP Reporters for Cells within the Osteoblastic Lineage.</a> J.Orthop.Res. 28 (10), 1338-1347 (2010).</li><li>Matthews, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+asMA-labeled+progenitor+cell+commitment+identifies+notch+signaling+as+an+important+pathway+in+fracture+healing.">Analysis of asMA-labeled progenitor cell commitment identifies notch signaling as an important pathway in fracture healing.</a> J.Bone Miner.Res. 29 (5), 1283-1294 (2014).</li><li>Dyment, N. A., Hagiwara, Y., Jiang, X., Huang, J., Adams, D. J., Rowe, 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=Response+of+knee+fibrocartilage+to+joint+destabilization.">Response of knee fibrocartilage to joint destabilization.</a> Osteoarthritis Cartilage. 23 (6), 996-1006 (2015).</li><li>Grcevic, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+fate+mapping+identifies+mesenchymal+progenitor+cells.">In vivo fate mapping identifies mesenchymal progenitor cells.</a> Stem Cells. 30 (2), 187-196 (2012).</li><li>Hong, S. H., Jiang, X., Chen, L., Josh, P., Shin, D. G., Rowe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computer-Automated+Static,+Dynamic+and+Cellular+Bone+Histomorphometry.">Computer-Automated Static, Dynamic and Cellular Bone Histomorphometry.</a> J Tissue Sci Eng. Suppl 1, 004 (2012).</li><li>Matthews, B. G., Torreggiani, E., Roeder, E., Matic, I., Grcevic, D., Kalajzic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteogenic+potential+of+alpha+smooth+muscle+actin+expressing+muscle+resident+progenitor+cells.">Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.</a> Bone. 84, 69-77 (2016).</li><li>Hagiwara, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fixation+stability+dictates+the+differentiation+pathway+of+periosteal+progenitor+cells+in+fracture+repair.">Fixation stability dictates the differentiation pathway of periosteal progenitor cells in fracture repair.</a> J.Orthop.Res. 33 (7), 948-956 (2015).</li><li>Jiang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+GFP+expression+in+murine+bone.">Histological analysis of GFP expression in murine bone.</a> J.Histochem.Cytochem. 53 (5), 593-602 (2005).</li><li>Nissanov, J., Bertrand, L., Tretiak, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryosectioning+distortion+reduction+using+tape+support.">Cryosectioning distortion reduction using tape support.</a> Microsc.Res.Tech. 53 (3), 239-240 (2001).</li><li>Kawamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+new+adhesive+film+for+the+preparation+of+multi-purpose+fresh-frozen+sections+from+hard+tissues,+whole-animals,+insects+and+plants.">Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants.</a> Arch.Histol.Cytol. 66 (2), 123-143 (2003).</li></ol>

The authors declare that they have no competing financial interests.

这里，我们已经提出了详细cryohistology协议共定位并通过在一个单一的部对准来自多轮染色/成像的图像量化几个生物措施。因为它维持的困难部分组织的形态（ 例如 ，矿化骨和软骨）使用cryotape概述的方法是特别有用的。此外，该截面​​组织被牢固地附着于玻璃载片，允许多轮同一节染色/成像的;不像在那里连续切片分别染色，不同的协议传统的方法。采用连续切片会带来一个问题，尝试将部分之间的共定位信号，东西是不是已经染色，并用多轮成像单部分问题时。 市场上的第一带稳定组织切片的产物是磁带传送系统16，17，它使用塑料带涂覆与被放置在组织冷冻块的表面上的冷温度粘合剂。当带下方的低温恒温器刀片割伤，该部分被去除完整并附着到磁带上。随后，胶带被置于样品面朝下上涂覆有紫外线固化胶，使得组织变得刚性连接到滑动幻灯片。一旦磁带产生了分离从部分远，滑动可以为任一荧光或发色组织学处理。粘合剂的背景荧光低，并且多轮染色和成像与大多数软组织效果很好。然而与矿化部分，从粘合带转移组织时向载玻片可以有矿物片段的损失。此外，它是一种相对昂贵的系统在低温恒温器（&gt; $ 8,000个）和耗材成本来安装被高（&gt; $ 2 /滑动）。 通过Tadafumi川本博士开发的另一个磁带稳定的战略使用了涂有粘合剂的聚偏二氯乙烯膜来捕获所述组织部分。在他们的协议中，新鲜冰冻组织切片在磁带上，并立即固定于PFA或乙醇18。接着，磁带染色，然后安装在与样品面朝下镜检载玻片。因此，每个染色协议上的不同录音部分进行。 我们已经修改，并加入到川本协议在许多方面，包括嵌入事先固定在福尔马林样品。这个步骤是关键的固定细胞的范围内的转基因动物的可溶性胞质GFP。我们已用于广泛的组织类型5-13的cryotape。实验室人员有限的组织经验，可以生产出高品质的部分用这种方法。该协议的主要优点是相对低的成本（无特殊仪器，&lt;每张幻灯片$ 1.00），无矿物片段的损失，并有能力在同一节进行多轮染色和成像。 因为在重复成像的滑动多次的相同区域将是不合理的大量甚至使用机动阶段幻灯片，这个协议的另一关键步骤是滑盖扫描显微镜来增加一致性和吞吐量的利用率。显微镜是，这两个表面荧光和透射光下操作的数字滑动扫描器。它配有一个10位机动炮塔和两个黑白和彩色摄像机。该系统的真正新颖性，在我们手中，是感兴趣的特定区域可被用户方便快捷地定义或由成像软件自动检测。这些感兴趣的区域然后可从第一轮成像保存，并在随后的几轮然后迅速重新装载。因此，所关注的相同区每轮成像期间成像。基于用户可在软件中定义的参数，在显微镜将自动对焦和扫描所感兴趣的区域内采集期间缝合平铺图像。 通过该用户执行多轮染色的顺序是重要的。一般顺序在图1中概述的。可通过荧光显微镜被充分地分开的荧光团的数目是的，可以在一个给定的部分中获取的应对措施的数量的主要限制因素。然而，荧光通道可以在某些情况下被重新使用。例如钙黄绿素蓝和去甲金霉素都发出黄色通道一个强烈的信号用来测量TRAP活性。这泄流通过避免，但因为告警缓冲区decalcifies的部分，从而去除到成像TRAP活动之前的矿物。此外，DAPI染液将在黄道发射也是如此。然而，用户可以在第另一染液代替DAPIe红色或远红光范围。此外，抗体可以被剥离，并与使用相同的荧光通道不同抗体重新染色。因此，这种方法是相当适合于增加可记录在一给定段的应对措施的数量。 存在与所呈现的协议的某些限制。 1）cryotape可能无法充分粘附到某些组织。例如，福尔马林固定的脑切片倾向于多轮染色后脱落磁带。对组织如此，因为组织是通过紫外线活化粘合剂（ 见表2为这两种方法之间的比较）加入了玻璃表面上的磁带传送系统可能更适合。 2）脱乙酰壳多糖粘合剂，同时提供透明的光学特性，将无法生存，包括高温或强酸苛刻的处理步骤。因此，在UV活化的粘合剂是更适合于这些应用程序（见表1纤维或者这两种粘合剂之间的比较）。 这里介绍的cryohistological协议还借给自己更高的吞吐量和自动化。例如，由cryotape提供的磁带稳定允许相对新手用户在相同的块切割复骨骼或关节一次，显著增加吞吐量。用自动扫描仪显著减少相关的成像，这通常是在我们的经验中的限速步骤的技术人员的时间和成本。能够稳定，可靠的图像的感兴趣几次同一区域在很短的一段时间内提供了自动化的，组织的客观分析的平台。事实上，我们的小组开发了计算机自动校准和骨形态计量学的一个平台。首先，软件对齐多轮基于所述荧光基准标记的图像。然后，将对准的图像被馈送到一形态计量学管道，其中COMPUT呃软件定义感兴趣的区域，客观地衡量几个静态和动态测量（多见于 www.bonebase.org）13。该平台是因为增加的吞吐量和一致性的cryotape切片，数字切片扫描显微镜和自定义分析软件提供了实现的。该协议代表在高通量cryohistology一个显著进步，并应使用这些成像难以部组织如骨，以及那些没有经验与组织切片。

生物学研究通常需要高效率的表型，这是经常用某种组织学分析1-3相关联。这需要的是更为明显的雄心勃勃的计划，扰乱小鼠基因组中4每个基因。这些组织学分析的范围可以从评估细胞形态和/或解剖特征来特定基因或蛋白质到单个细胞的映射表达式。实际上，组织学的基因组学领域中的基本贡献之一是为特定的分子信号到特定区域或细胞类型相关联的能力。 组织学的传统方法，尤其是对肌肉骨骼组织中，往往费时费力，有时需要数周来修复时间，脱钙，部分染色和图像标本然后分析通过人眼可识别图像。分析多个分子信号，通过免疫组化，是否在原地 hybridizat离子或特殊污渍，需要多个章节，甚至多个标本适当地执行。此外，这些多个响应无法被共定位到相同的细胞，有时不能共定位到特定的区域的给定样品中。随着基因组学和表观基因组学领域的移动进入数字化时代，组织学领域也必须跟进，提供高效，高通量，以及单个组织切片中的各种分子信号的自动分析。 事实上，存在对可以在给定样品中的特定小区中的多个分子信号关联改进的组织学技术的需求。最近，我们已经发布了从矿化组织5-14评估某一部分内的几个应对措施的新高通量方法cryohistological。该过程涉及稳定冷冻cryotape的冷冻切片，拘泥于所录部分显微镜幻灯片，开展几轮染色和成像上的每节。这些轮次的图像，然后手动或通过之前的图像分析计算机自动化（ 图1）对齐。在这里，我们提出这一过程的详细协议的，并提供这些技术提高了我们不同的生物过程的理解的例子。

<table border="0" cellpadding="0" cellspacing="0" width="1066">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">10% neutral buffered formalin</td>
			<td style="width:185px;">Sigma Aldrich</td>
			<td style="width:259px;">HT501128-4L</td>
			<td style="width:359px;">Multiple suppliers available. Toxic. Can be substituted with 4% paraformaldehyde.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Sigma Aldrich</td>
			<td>P5368</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomolds</td>
			<td>Fisher Scientific</td>
			<td>Fisherbrand #22-363-554</td>
			<td>Different sizes can be used depending on tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomatrix</td>
			<td>Thermo Scientific</td>
			<td>6769006</td>
			<td>Can be substitituted with other cryomatrices. However, PVA/PEG-based resins have worked best in our hands.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-methyl-butane</td>
			<td>Sigma Aldrich</td>
			<td>M32631</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat</td>
			<td>Leica Biosystems</td>
			<td>3050s</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Specimen disc</td>
			<td>Leica Biosystems</td>
			<td>14037008587</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat blades</td>
			<td>Thermo Scientific</td>
			<td>3051835</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryotape</td>
			<td>Section Lab</td>
			<td>Cryofilm 2C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-46</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic microscope slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>71890-01</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass microscope slides</td>
			<td>Thermo Scientific</td>
			<td>3051</td>
			<td>Can be substituted with other brands/models.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Norland Optical Adhesive, 61</td>
			<td>Norland Optical</td>
			<td>Norland Optical Adhesive, 61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UV Black Light</td>
			<td>General Electric</td>
			<td>F15T8-BLB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>ARK2183</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chitosan</td>
			<td>Sigma Aldrich</td>
			<td>C3646</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InSpeck red microscopheres</td>
			<td>ThermoFisher Scientific</td>
			<td>I-14787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InSpeck green microspheres</td>
			<td>ThermoFisher Scientific</td>
			<td>I-14785</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcein Blue</td>
			<td>Sigma Aldrich</td>
			<td>M1255</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcein</td>
			<td>Sigma Aldrich</td>
			<td>C0875&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alizarin complexone</td>
			<td>Sigma Aldrich</td>
			<td>A3882&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Demeclocycline</td>
			<td>Sigma Aldrich</td>
			<td>D6140&#160;</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G5516</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ELF 97 yellow fluorescent acid phosphatase substrate</td>
			<td>ThermoFisher Scientific</td>
			<td>E-6588</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>62247</td>
			<td>Multiple suppliers available. Can be substituted with Hoechst 33342 or other nuclear dyes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TO-PRO-3 (Cy5 nuclear counterstain)</td>
			<td>ThermoFisher Scientific</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propidium Iodide</td>
			<td>ThermoFisher Scientific</td>
			<td>R37108</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium acetate anhydrous</td>
			<td>Sigma Aldrich</td>
			<td>S2889</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium tartrate dibasic dihydrate&#160;</td>
			<td>Sigma Aldrich</td>
			<td>T6521</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium nitrite</td>
			<td>Sigma Aldrich</td>
			<td>S2252</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Sigma Aldrich</td>
			<td>15504</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2 hexahydrate</td>
			<td>Sigma Aldrich</td>
			<td>M9272</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich</td>
			<td>S7653</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast Red TR Salt</td>
			<td>Sigma Aldrich</td>
			<td>F8764</td>
			<td>Multiple suppliers available. Can also be substituted with other substrate kits such as Vector Blue (Vector Laboratories, Cat# SK-5300)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Naphthol AS-MX&#160;</td>
			<td>Sigma Aldrich</td>
			<td>N4875</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N dimethylformamide</td>
			<td>Sigma Aldrich</td>
			<td>D158550</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Toluidine blue O</td>
			<td>Sigma Aldrich</td>
			<td>T3260</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Axio Scan.Z1</td>
			<td>Carl Zeiss AG</td>
			<td>Axio Scan.Z1</td>
			<td>Other linear or tiled scanners may also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">YFP Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom yellow (ELF 97) Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>custom; HQ409sp, HQ555/30m, 425dxcr</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRITC Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cy5 Filter Set</td>
			<td>Chroma Technology Corp.</td>
			<td>49009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Tape Transfer System</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-10</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Tape Windows</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-72</td>
			<td>Multiple suppliers available.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CryoJane Adhesive Slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>62800-4X</td>
			<td>Multiple suppliers available.</td>
		</tr>
	</tbody>
</table>

high-throughput, multi-image cryohistology of mineralized tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

的一般工作流程为高通量，多图像Cryohistology 图1表示用于本技术的一般的工作流程。它包括从固定经过几轮的成像和图像终于对准/分析的几个步骤。该过程可能需要少至一个星期来从样品固定通过4轮成像的，这是比需要脱钙这些类型的样品更少的时间去。成像的顺序通常开始与已经在检体（ 例如 ，两性平等问题协调，矿化标签等 ），然后复用荧光免疫染色，随后用荧光酶活性测定法（ 例如 ，TRAP，AP 等 ）的内源信号和细胞周期分析测定法（ 例如 ，EDU）和最后与生色染色完成（ 例如 ，甲苯胺蓝0，下摆atoxylin，番红O 等 ）。 从少年踝关节 6 代表性的例子 这个特殊研究的目的是为了展示不同类型的胶原蛋白（ 即 COL1A1， 的Col2a1，和COL10A1）与跟腱-骨插入位点（ 即附着点）的纤维软骨的矿化表达之间的关系。因此，三重转基因荧光报道小鼠包括COL1A1-GFPTpz， 的Col2a1-CFP和COL10A1-mcherry用于鉴定表达各转基因的细胞。第一轮成像是内源转基因表达的从两周龄小鼠，其对应于当在跟腱（ 图4B）的附着点矿化。第二轮成像然后对进行多光子显微镜经由两个光子的二次谐波产生（SHG， 图4C）获得的胶原结构的图像。此步骤中使用的附着点内的胶原纤维的碱，以确定细胞。第三轮成像是印度刺猬（IHH），这是促进附着点（ 图4D）的矿化的主要信令配体之一的免疫染色步骤。第四轮成像是使用蓝色碱性磷酸酶底物试剂盒，其引起既Cy5的荧光和蓝色的信号，进行可视化活性矿物沉积（ 图4E）的区域AP染色。最后，第五轮的成像是TB染色可视化的解剖学特征，包括纤维软骨（ 图4F）内的蛋白多糖含量。所有的图像手动图像编辑软件中的对齐。在五轮成像是在4天时间，其中3跟着进行来样加工及切片（共7天）的天。 从成人膝关节 11 代表性的例子 该实验的目的是确定发生在膝的内侧副韧带（MCL）的下列经由前十字韧带（ACL）的横断关节不稳定的附着点的矿化的变化。矿化的变化可以在MCL附着点，早在两个星期手术后这3个月大的老鼠可以看出。要监视附着点内的纤维软骨矿化沉积，矿物标签是在2周手术后给予手术（去甲金霉素）的一天，老鼠和前牺牲（钙黄绿素）的一天。老鼠还包括COL1A1-CFP和COL10A1，mcherry荧光记者，监测矿化和矿化fibrochondrocyt的胶原表达ES，分别。第一轮成像是内源信号，在这种情况下对应于荧光蛋白和荧光矿化标签（ 图5A）的。第二轮包括TRAP染色以展示下面的骨髓（ 图5B）这种酶在矿化纤维软骨细胞中的附着点的表达以及破骨细胞。第三轮是AP染色证明纤维软骨细胞的活性矿化以及下面的骨骼（ 图5C）的成骨细胞的区域。最后，TB染色第四轮（ 图5D）进行。所有的图像手动图像编辑软件中的对齐。再次从组织收获带到图像对准的总时间为7天。 从股骨远端骨小梁的代表性的例子 <p class="jove_content" fo:kee对 - together.within页="1"&gt;本研究的目的是通过进行自动动态组织形态计量学（www.bonebase.org）具有不同遗传背景的表型的小鼠的骨架。三个月大的小鼠，给出了两个矿化标签（牺牲前钙黄绿7天，茜素络合处死前2天）。从四个独立的小鼠股骨远端分别嵌入同一冰冻块中再砍在一起。包含四个骨骼部分被粘到的幻灯片。接着，含有4个额外的骨骼另一部分被粘向下相邻的第一部分。因此，总共8骨头被应用到每个滑板（ 图3B）。这个过程对女8和在骨髓内3个不同层次8雄性小鼠进行，得到12幻灯片的总数。基准标记（微球）的骨骺并在干燥部（ 图3C）的中间骨干内施加。全部12张幻灯片染色FOř钙黄绿素蓝和第一轮成像（ 图6A）的过程中成像为积累矿物（钙黄绿素蓝）和矿化标签（钙黄绿素和茜素配位）。然后将载玻片成像为在第三轮成像的第二轮和AP活性（ 图6C）TRAP活性（ 图6B）。最后，载玻片用在第四轮（ 图6D）甲苯胺蓝染色。基准标记每一成像轮，包括色轮期间进行成像，并使用定制软件进行比对。以提供通过此过程，32骨头（16股骨和16椎体）包埋在8个块，3个部分，从每个块所采取的一个想法，将切片在12个载玻片分布，和12载玻片成像4次96生产合成图像堆栈。执行此实验的总时间为8天。 图1.典型的工作流的协议。一般步骤包括:1）组织固定，2）带稳定cryosectioning，3）录音部分坚持载玻片，4）参考标记应用，5）染色的多轮和6）成像和7）的图像组装，调整和分析。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54468/54468fig2.jpg" /> 图2. 高通量嵌入和磁带稳定Cryosectioning，由于cryotape提供了稳定的，多个骨骼可以嵌入彼此相邻（AB）及切片同时（CD）。一块干冰的过程中使用嵌入过程拘泥于固定到位的骨头冻结整个冷冻块（B）前。该cryotape被卷到块（C）和部分仍然切片（D）的时候卡住磁 ​​带。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54468/54468fig3.jpg" /> 图3. 第录播载玻片，参考标记的应用，和幻灯片装载到幻灯片扫描显微镜托盘的坚持。录音的部分粘到与任一UV固化或壳聚糖基胶粘剂载玻片的表面（A ）。以下固化或干燥，只差，粘合剂的平坦层保持cryotape和玻璃表面（B）之间。参考三​​月KERS被应用到干片（C，箭头指向掉落微球溶液）。然后幻灯片安装在显微镜盘（D），并装入显微镜（E）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54468/54468fig4.jpg" /> 图4.从二周龄鼠标跟腱成像五个回合。复合图像堆栈（A）从五轮成像创建。第1轮（B）:内源性COL1A1-GFPTpz， 的Col2a1-CFP和COL10A1，mcherry转基因的表达。在双光子显微镜胶原二次谐波产生（SHG）:轮2（℃）。第3轮（D）:免疫染色IHH。第4轮（E）:AP酶活性。第5轮:甲苯胺蓝O（F）。这个数字已经从Dyment 等修改，2015年6。比例尺= 200微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54468/54468fig5.jpg" /> 图5. MCL附着点的成像四轮以下联合不稳定。跪在三个月大的小鼠不稳定，从而增加了MCL附着点的矿化。一个地美环素矿物标签被赋予手术和矿物质钙黄绿素标签的一天给牺牲的前一天。第1轮（A）:内源性COL1A1-CFP，拥有地美环素和钙黄绿矿物标签COL10A1-mcherry转基因的表达。第2轮（B）&lt;/ STRONG&gt;:TRAP酶活性。轮3（C）:AP酶活性。第4轮（D）:甲苯胺蓝O.陷阱以及AP信号覆盖于公元前面板TB信号之上。黄色通道可以再次因为告警缓冲区decalcifies组织，消除地美环素标签可用于陷阱。这个数字已经从Dyment 等修改，2015年11。比例尺= 200微米。民主党:地美环素，TRAP:抗酒石酸酸性磷酸酶，AP:碱性磷酸酶，MCL:内侧副韧带。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/54468/54468fig6.jpg" /> 图6. 股骨远端内四轮成像的含微小参考标记。三个月之久的话筒Ë给予钙黄绿素和茜素络合矿物标签回合1（AA&#39;）:除了内源性钙黄绿素和alizarain络合标签钙黄绿素积累矿物蓝染色第2轮 （BB&#39;）:TRAP酶活性第3轮 （CC&#39;）。 :AP酶活性第4轮 （DD&#39;）:甲苯胺蓝O.绿色微球在每个回合成像（A&#39;-D&#39;，箭头表示在所有图像相同的微球）。比例尺= 200微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54468/54468fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td>壳聚糖胶粘剂</td><td> UV固化胶</td></tr><tr><td>胶粘剂机制</td><td>蒸发</td><td> UV聚合</td></tr><tr><td>固化时间</td><td> &gt; 24小时</td><td> &lt;20分钟</td></tr><tr><td>可以部分粘合剂固化后会被删除？ </td><td>是</td><td>没有</td></tr><tr><td>固化胶粘剂可溶性？ </td><td>是的，在低pH值酸性溶液</td><td>没有</td></tr><tr><td>是否胶粘剂承受热抗原修复？ </td><td>没有</td><td>是</td></tr><tr><td>胶粘剂是自体荧光？ </td><td>没有</td><td>在最小的范围内紫外线</td></tr></tbody></table> 表1.壳聚糖胶粘剂和UV固化胶粘剂的比较 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> Cryotape </td><td>磁带传输系统</td></tr><tr><td>可以使用这个系统部分矿化骨？ </td><td>是</td><td>是的，但片矿化骨可能不能完全转移到滑动</td></tr><tr><td>可以使用这个系统部分矿化关节？ </td><td>是</td><td>是</td></tr><tr><td>可以使用该系统部分的大脑？ </td><td>是的，但可以组织多轮成像后脱落胶带</td><td>是</td></tr><tr><td>可能减少嵌入在同一块多个样品？ </td><td>是</td><td>是</td></tr><tr><td>可能在同一节进行多轮影像？ </td><td>是</td><td>是</td></tr></tbody></table> 表2. Cryotape系统和磁带传输系统的比较

Watch this Scientific Journal Video about High-Throughput, Multi-Image Cryohistology of Mineralized Tissues at JoVE.com

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

high-throughput-multi-image-cryohistology-of-mineralized-tissues

Nanyang Technological University

Research

JoVE Journal

Biology

15.8K 次观看.  University of Connecticut Health Center. In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues. 

视频: High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

科研

生物学

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

木质纤维素材料的高通量检测糖化

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

高通量的亲和标签的重组蛋白的纯化

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

海胆受精卵的高通量显微注射

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

萤光素酶表达的重组病毒的高通量滴定

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

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 ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

激酶底物对鉴定使用高通量筛选


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

高通量<em&gt;斑马鱼</em&gt;能量消耗分析

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...

Precise, High-throughput Analysis of Bacterial Growth

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the systems level. Recent studies have reported correlations between bacterial growth and genome-wide events, such as genome reduction and transcriptome reorganization. Correctly analyzing bacterial growth is crucial for understanding the growth-dependent coordination of gene functions and cellular components. Accordingly, the precise quantitative evaluation of bacterial growth in a high-throughput manner is required. Emerging technological developments offer new experimental tools that allow updates of the methods used for studying bacterial growth. The protocol introduced here employs a microplate reader with a highly optimized experimental procedure for the reproducible and precise evaluation of bacterial growth. This protocol was used to evaluate the growth of several previously described Escherichia coli strains. The main steps of the protocol are as follows: the preparation of a large number of cell stocks in small vials for repeated tests with reproducible results, the use of 96-well plates for high-throughput growth evaluation, and the manual calculation of two major parameters (i.e., maximal growth rate and population density) representing the growth dynamics. In comparison to the traditional colony-forming unit (CFU) assay, which counts the cells that are cultured in glass tubes over time on agar plates, the present method is more efficient and provides more detailed temporal records of growth changes, but has a stricter detection limit at low population densities. In summary, the described method is advantageous for the precise and reproducible high-throughput analysis of bacterial growth, which can be used to draw conceptual conclusions or to make theoretical observations.

Quantitative evaluation of bacterial growth is essential to understanding microbial physiology as a systems-level phenomenon. A protocol for experimental manipulation and an analytical approach are introduced, allowing for precise, high-throughput analysis of bacterial growth, which is a key subject of interest in systems biology.

细菌生长的精确、高通量分析

Bacterial growth is a central concept in the development of modern microbial physiology, as well as in the investigation of cellular dynamics at the ...