作者想感谢他持续的技术支持Ikiel Ptito。 NSERC授予到MP。

第1部分：前处理的组织应根据伯克等。 （2009）5。 <ol><li>简单地说，组织应该得到很好的多聚甲醛，戊二醛，或福尔马林灌注。这可以通过标准transcardial，通常用于收获其他器官灌注。在本研究的主题是深刻的盐酸氯胺酮（10毫克/公斤，IM）与戊巴比妥钠（25毫克/公斤，四）过量安乐死，镇静剂和用0.1 M PBS灌注，直到完全exsanguinated transcardially人。其次是在PBS的4％多聚甲醛为5分钟（约1升）的解决方案。组织应paraformaldeyhde，gluteraldehyde，或福尔马林灌注。大脑应stereotaxically封锁，从头骨取出，称重，量确定，cryoprotected，并冻结5。 </li></ol>第2部分：组织系统抽样，根据伯克等。 （2009）6。 <ol><li>在这个例子中，我们将重点放在vervet脑额叶。参考空间被定义为包括左半球额极皮层组织从中央沟。该系列对于我们的目的是在整个大脑皮层的1 / 10节，在50μm的切片。一个完整的系列染色甲酚紫（＃1系列）。系列2分裂，因此，有一半的路段髓鞘的启示和乙酰胆碱酯酶的另一半染（划定一定的皮层下区）与氯化金染色。所有其他系列抗原保存，作为我们长期的研究计划的一部分库存。 </li></ol>第3部分：视学（更多详情，请参阅2002年木桐） 4 <ol><li> （N）的手机号码，总的估计是基于下列公式计算： N = SSF -1 x ASF -1 x TSF -1 xΣQ - SSF是部分抽样比，ASF是该地区的抽样比例，TSF，如果厚度采样的分数（测量组织厚度划分是由剥离高度），ΣQ -计算利息的对象总数内剥离。此协议的下一个部分显示了如何确定的社保基金理事会，ASF，TSF和 Σ Q。 </li><li>第抽样比（非电脑为基础的） <ol style="list-style-type:lower-alpha;"><li>完整的参考空间，必须明确界定，由前到后和背腹。在这个例子中，我们在额叶的兴趣，并定义为额极（前）的一角，以中央沟（后）和外侧沟（腹）地区，并排除岛。 </li><li>对于这一特定主题的额叶，共760节，有系统地搜集76甲酚紫染色。由于目标是10个部分，整个参考空间的1 / 6甲酚紫染色切片取样，导致了大约1 / 60节中的示例分数。我们随机开始的第6甲酚紫染色部分之一和系统采样此后的1 / 6。一个13节，共抽样选定的主题（图2）。 </li></ol></li><li> 区域样本分数（计算机为基础的） <ol style="list-style-type:lower-alpha;"><li>的试验研究表明最佳网格大小，区的抽样比例，约100-300 disectors样品的参考空间。在额叶，网格尺寸为2500微米2取得 ​​了平均254 disectors这个问题（图2）。 disector的大小，产量约0-5计算对象，在这种情况下，神经元之间。 </li></ol></li><li>厚度样品组分（以计算机为基础） <ol style="list-style-type:lower-alpha;"><li>在每个disector位置，测量组织的高度，这个高度的一小部分进行采样，这是已知厚度抽样比。要确定测量的厚度，重点通过z平面，直到第一个单元格来成为关注的焦点，然后备份轻微的部分，即顶部，直到出现的最后一个对象只是重点。要确定组织的底部，重点是通过z平面，直到细胞失焦勉强。 </li><li>通过z平面为重点，细胞应在每一个深度染色，如果没有，这可能表明不完整的组织染色或非均匀脱水渗透，在这种情况下，部分应重新染色。这种预防措施是特别重要的免疫染色的组织，需要通过比较厚的组织切片immunoprobes渗透的。出于这个原因，免疫染色组织应轻轻counterstained嗜碱性染色（例如，甲酚紫，赫马toxylin）为了准确地判断顶部和底部的部分，并确认抗体的渗透。在这项研究中，部分在50μm的切片设置切片。毕竟组织处理完成后，测量组织厚度平均为17.9μm（图2），用了约65％的平均收缩。请注意，这些结果代表了典型的收缩处理组织常规病理准备 7 。 </li><li>默认为典型的研究disector高度为10μm。 disector高度和测量的部分厚度之间的区别是后卫身高，在没有生物的特点是计算量组织。卫兵高度的使用，避免在切片表面组织损伤（如丢失上限）。 </li></ol></li><li>对象总数计算，ΣQ - <ol style="list-style-type:lower-alpha;"><li>为了避免出现识别错误的偏见，这是一个标准的定义必须是特别感兴趣的生物特征。在这种情况下，我们感兴趣的计数神经元。因此，一个神经元被定义为一个位于市中心的可见核仁和细胞质明确界定，而神经胶质细胞普遍缺乏可见核仁和细胞质。对于这个问题的457神经元进行计数。 </li></ol></li></ol>第4部分：Stereologer -一个计算机化的体视系统（体视学资源中心，切斯特，MD） <ol><li> Stereologer系统提示用户填写必要的信息在一步一步的时尚。在“研究资料”一节中的参数确定的研究。由于参考空间必须被定义为研究对象，应选择体积参数（参考空间卡瓦列利估计应选择）。物体的体积也可以在此处选择估计数加权感兴趣的对象的人口数量。对于我们的例子中，只有量的参考空间被选中。对于每个对象，选择的数量和定义功能，在这种情况下，神经元的利息。 </li><li>案例初始化：是建立在本节中的抽样信息。输入板的采样间隔（如果单独组织切片详尽砖，每节按顺序放在然后请在这里输入1）。通过参考空间（在这种情况下，760），采取部分输入的总数。然后输入部分的取样时间间隔（在这种情况下，59）。然后，系统计算进行采样的部分（在这种情况下，13）。 </li><li>探头参数：本节定义了电网和disector大小。对于体积，使用低倍率2.5倍，10倍编辑“菜单下的定义网格间距。对象的放大倍数应在100X（NA 1.3或1.4）。帧面积disector的大小，在这种情况下，我们用50％的屏幕。帧高度disector厚度在10微米这里设置。帧间距网格的大小。在我们的试验研究中，我们发现，2500μm产量在150-200帧之间的间隔一致的方式通过一个比较大的参考空间的系统。对于规模较小的地区，应该使用较小的网格大小。的试验研究，确定为每个特定的研究的光学探头参数。 </li><li>一旦已经建立了研究参数，通过组织取样进行。程序将提示您样本第一部分第1步：低倍率下，程序会提示用户，跟踪上一节中的参考空间。然后系统将探头上的参数和用户基础的部分放在一个网格，然后将验证点内的参考空间。如果一个点是不是在参考空间点上只需点击并不会被计算入量第2步：系统将在参考空间放置一个新的网格，基于帧间隔参数。验证参考空间的交点属于第三步：然后系统会提示用户切换到高放大倍率的目标和移动舞台，第一次剥离第4步：此时系统会提示用户定义部分的顶部和底部，然后，系统设置采样空间， 在Z轴第 5步：用户将落在disector通过z平面内的每个对象，然后在。触碰红线或disector底部的对象，可能不被计算在内。一旦计算每个对象，然后单击下一步。该阶段将移动到下一个disector和重复4和5的步骤。一旦节disectors都被计算在内，系统会提示用户插入下一个连续的部分，被探测。将重复步骤1-5，直到所有的顺序部分被采样。该系统将提供ASF，SSF，TSF，和ΣQ计算。根据这些参数，系统将产生估计Ñ，参考量，估计的数量和体积的行政长官。它还将变得更有效率的建议或减少行政长官（图2） 。 </li></ol>第5部分：代表性的成果： 无偏体视学提供了参考空间内细胞群的有效和可靠的估计。有计算机为基础的体视系统，所有这些都依赖系统抽样和定义的参考空间。我们使用BioQuant生命科学系统的估计2岁vervets的大脑皮层神经元的总人口将​​超过828万（图3）与0.042的平均行政长官。我们随后使用Stereologer系统估计，额叶皮层神经元的一半左右8号帐户。 <img src="/files/ftp_upload/1262/1262fig1.jpg" alt="图1" /> 图1基于计算机的体视系统。一套基本的体视系统是相同的，在显微镜与机动阶段（XYZ），一个阶段控制器，摄像机，电脑，和软件。最终选择的系统应根据效率，需要和成本分析。 <div style="border:1px #000000 solid"><table border="0"><tbody><tr><td colspan="2">研究资料</td></tr><tr><td>研究名称</td><td>额叶皮质</td></tr><tr><td>首席研究员</td><td> MB </td></tr><tr><td>物种</td><td>公司</td></tr><tr><td>参考空间前腰</td><td>皮质</td></tr><tr><td colspan="2">注释</td></tr><tr><td colspan="2">案件信息</td></tr><tr><td>数据采集​​器</td><td> MB </td></tr><tr><td>日期</td><td> ，2008年4月30日，日（星期三） </td></tr><tr><td>集团</td><td>控制</td></tr><tr><td>主题</td><td> O26 </td></tr><tr><td colspan="2">注释</td></tr><tr><td colspan="2">采样特性</td></tr><tr><td>板坯采样间隔</td><td> 1 </td></tr><tr><td>各组的总人数</td><td> 760 </td></tr><tr><td>部分的取样时间间隔</td><td> 59 </td></tr><tr><td>开始第</td><td> 2 </td></tr><tr><td colspan="2">馏分</td></tr><tr><td>业余</td><td> 0.0002 </td></tr><tr><td> SlabSF </td><td> 1.0000 </td></tr><tr><td>社保基金理事会</td><td> 0.0169 </td></tr><tr><td> TSF </td><td> 0.5587 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="6">业绩摘要</td></tr><tr><td>参数</td><td>探头</td><td>名称</td><td>结果</td><td>行政长官</td><td> SD </td></tr><tr><td>厚度</td><td> --- </td><td> --- </td><td> 17.8996μ </td><td> --- </td><td> --- </td></tr><tr><td>数</td><td> Disector </td><td>神经元</td><td> 243833769.1473 </td><td> 0.0474 </td><td> N / A </td></tr><tr><td>卷</td><td>卡瓦列利点网格</td><td>体积</td><td> 1719769397954.1284μ^ 3 </td><td> 0.0078 </td><td> N / A </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="2">建议</td></tr><tr><td>探头</td><td>对象编号（神经元） </td></tr><tr><td>行政长官</td><td> 0.0474 </td></tr><tr><td>建议</td><td>行政长官是可以接受的。 </td></tr></tbody></表&gt; <table border="0"><tbody><tr><td colspan="2">观看Disectors数过高，增加Disector间距。 </td></tr><tr><td>探头</td><td>区卷（卷） </td></tr><tr><td>行政长官</td><td> 0.0078 </td></tr><tr><td>建议</td><td>行政长官是可以接受的。 </td></tr></tbody></table><table border="0"><tbody><tr><td colspan="3">部分结果</td></tr><tr><td>第</td><td>神经元NumObjects神经NumCorners </td><td>体积RegPoints </td></tr><tr><td> 1。 40 </td><td> 48 </td><td> 34 </td></tr><tr><td> 2。 26 </td><td> 52 </td><td> 44 </td></tr><tr><td> 3。 29 </td><td> 56 </td><td> 48 </td></tr><tr><td> 4。 39 </td><td> 100 </td><td> 83 </td></tr><tr><td> 5。 49 </td><td> 112 </td><td> 83 </td></tr><tr><td> 6。 53 </td><td> 156 </td><td> 120 </td></tr><tr><td> 7。 65 </td><td> 128 </td><td> 106 </td></tr><tr><td> 8。 45 </td><td> 108 </td><td> 100 </td></tr><tr><td> 9。 52 </td><td> 100 </td><td> 77 </td></tr><tr><td> 10。 26 </td><td> 64 </td><td> 60 </td></tr><tr><td> 11。 21 </td><td> 44 </td><td> 44 </td></tr><tr><td> 12。 11 </td><td> 36 </td><td> 32 </td></tr><tr><td> 13。 1 </td><td> 12 </td><td> 6 </td></tr></tbody></table></div> 图2结果Stereologer系统取得的泉叶 ，额叶，系统抽样和切片的体视学过程中最长的部分。完成了地形，体积和神经元的估计，在大约一天每个科目。左半球是采样的数量是在两个半球的近似估计的文本增加了一倍。 <img src="/files/ftp_upload/1262/1262fig3.jpg" alt="图3" /> 图3皮层神经元的人口估计与BioQuant获得。BioQuant系统需要一个确切的地形之前，细胞计数。在这里，平均13节进行了采样。皮质地形和后续量估计为15-20小时之间，每个主题。地形下一个2.5倍的目标和计数使用100倍的油浸物镜（NA 1.3）。每第100节被选为整个平均180采样disectors左半球。一旦完成地形神经元计数了大约一天。左半球是采样的数量是在两个半球的近似估计的文本增加了一倍。

<ol>
	<li>West, M., Slomianka, L., Gundersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+the+subdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.">Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator.</a> Aat Rec. 231, 482-497 (1991).</li><li>Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+way+you+cut+it:+a+new+journal+policy+for+the+use+of+unbiased+counting+methods.">Any way you cut it: a new journal policy for the use of unbiased counting methods.</a> J Comp Neurol. 364, 5-5 (1996).</li><li>Sterio, D. 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+unbiased+estimation+of+number+and+sizes+of+arbitrary+particles+using+the+disector.">The unbiased estimation of number and sizes of arbitrary particles using the disector.</a> J Microsc. 134, 127-136 (1984).</li><li>Mouton, P. 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> Principles and Practices of Unbiased Stereology. , (2002).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+non-human+primate+brain+in+stereotaxic+space.">Dissecting the non-human primate brain in stereotaxic space.</a> J Vis Exp. 29, (2009).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+banking:+Making+the+most+of+your+research.">Brain banking: Making the most of your research.</a> J Vis Exp. 29, (2009).</li><li>Manaye, K. F., Wang, P., O'Neil, J., Huafu, S., Tizabi, Y., Thompson, N., Ottinger, M. A., Ingram, D. K., Mouton, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+Quantification+Of+Dtg+APP/PS1:+Neuroimaging,+Stereology,+And+Biochemistry.">Neuropathological Quantification Of Dtg APP/PS1: Neuroimaging, Stereology, And Biochemistry.</a> AGE. 29, 87-96 (2009).</li><li>Burke, M. W., Palmour, R. M., Ervin, F. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+reduction+in+frontal+cortex+of+primates+after+prenatal+alcohol+exposure.">Neuronal reduction in frontal cortex of primates after prenatal alcohol exposure.</a> Neuroreport. 20, 13-17 (2009).</li><li>Zangenehpour, S., Burke, M. W., Chaudhuri, A., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Batch+immunostaining+for+large-scale+protein+detection+in+the+whole+monkey+brain.">Batch immunostaining for large-scale protein detection in the whole monkey brain.</a> J Vis Exp. 29, (2009).</li></ol>

在灵长类动物的大脑无偏体视学与其它生物标本。然而，由于灵长类动物的大脑的大小，谨慎处理计划建议，因为一个单个半球的vervet猴子在50μm的切片时，产量超过1200节。首先，我们建议stereotaxically阻断大脑切成1cm的块，它提供了一个标准节块和学科之间的平面，最大限度地减少块之间的部分路段 ，并提供管理的5大小的块。一个关键的体视步骤是系统抽样6-10节获得通过参考区域。对?...

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<td>Stereologer</td>
<td>&nbsp;</td>
<td>Stereology Resource Center (Chester, MD)</td>
<td>www.disector.com</td>
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<td>Stereology Tool-Kit Stereologer system</td>
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<td>BioQuant Life Sciences (Nashville, TN)</td>
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<td>StereoInvestigator</td>
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<td>MicroBright Field (Willston, VT)</td>
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knowing what counts: unbiased stereology in the non-human primate brain

非人类灵长类动物是了解人类大脑的正常功能和疾病过程的一个重要平移物种。无偏体视学，接受国家的最先进的生物对象在组织切片进行量化的方法<sup&gt; 2</sup&gt;，生成可靠的结构数据，哺乳动物的大脑中的生物特征<sup&gt; 3</sup&gt;。该方法的关键组件是公正的（系统的，随机）解剖学定义的结构（参考空格）内细胞数量的大小，纤维和毛细血管长度，面积，区域卷和空间分布的生物对象的量化相结合，抽样参考空间<sup&gt; 4</sup&gt;。在前面的方法，这些体视学方法的优点是避免了所有已知的来源系统的（非随机）产生的错误，从错误的假设和非核查的模型。这项研究的文件一个计算机化的视学的生物应用vervet猴脑额叶皮层神经元的总人口估计（<em&gt; Chlorocebus aethiops sabeus</em&gt;），与两个市售体视节目，生命科学BioQuant和Stereologer（图1）的援助。此外对比和比较，从两者的BioQuant的结果和<em&gt; Stereologer</em“&gt;”系统“，这项研究提供了一个详细的协议<em&gt; Stereologer</em&gt;系统。

Watch this Scientific Journal Video about Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain at JoVE.com

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

在人体正常和病理条件下的灵长类动物的大脑解剖组织可以提供重要的见解。无偏体视学是一种准确，有效地估算总神经元的数量（或其他类型的细胞）在一个给定的参考空间的方法<sup&gt; 1</sup&gt;。

知道重要的：无偏体视学在非人类灵长类动物的脑

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

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JoVE Journal

Biology

13.6K 次观看.  University of Montreal. 在人体正常和病理条件下的灵长类动物的大脑解剖组织可以提供重要的见解。无偏体视学是一种准确，有效地估算总神经元的数量（或其他类型的细胞）在一个给定的参考空间的方法 1。

视频: Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

光固化牙科粘合树脂安装电极或微透析探针在慢性实验中的应用

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

科研

生物学

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

剖析在非人类灵长类动物脑立体定向空间

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

到大脑的门户：剖析灵长类动物的眼睛

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

测定哺乳动物细胞计数，细胞的大小和使用末席ž迷你自动细胞计数细胞的健康

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

生理学实验室演示：肾小球滤过率在一个老鼠

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

从大鼠的侧尾静脉血液取样

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo: 三维脂肪组织成像的组织清除方法

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

灵长类动物脸演变过程中的反向解剖与 dicect 显示其他隐藏数据

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...