室间隔新Yeshiva大学爱因斯坦医学院的研究者从启动资金支持。

1。动物灌注和小脑剖析<ol><li>根据蛋白质，灌注可能是必不可少的成功染色1,2。 transcardiac灌注是一种侵入性，非生存的过程，需要正确使用的麻醉药。正确的培训，机构批准，并IACUC批准，都在尝试的过程之前，必要的。它始终是一个好主意，咨询机构的兽医，以确定实验的要求，并获得正确的训练中得到的帮助。手术前，动物应该是深度麻醉，没有反应，如脚或尾巴捏刺激。用剪刀在腹部的皮肤和肌肉壁的切割，然后继续打开切割毗邻每侧胸骨肋骨。此外，实验者可以创造一个更大的内部工作空间切割筋膜周围的隔膜。提起腹部和胸壁superioRLY，心将予以曝光。做一个小切口在右心房，让血液流了出来，并立即插入左心室灌注针。一个基本的水压泵优先提供灌注的解决方案（ 详见表1）。首先，冲洗0.1M磷酸盐缓冲液（PBS， 见表3），直到流体运行明确血液。然后，关闭静压泵，快速切换的解决方案交付管的4％多聚甲醛的容器（PFA， 见表3）在PBS稀释，然后打开泵提供的固定液。泵的使用是没有必要的：与实践，填补两个注射器与PBS和4％的煤灰可以用来慢慢地提供解决方案。 </li><li>关键是要仔细解剖的头骨，脑，而不引入任何病变组织。即使小割伤，在解剖大脑会逐渐扩大成可见的缝隙，陷阱抗体，并导致artifactual染色处理的组织（如红色星号在图2）。小脑解剖，这是典型的，以减少中间的头部皮肤，然后轻轻地挑逗皮瓣除了暴露头骨。削减头骨由前至后沿背中线，还横向沿颅骨两侧。切不要超越前囟门运行的刻痕小脑的风险。使用镊子轻轻抬离头骨前，小心不要撕破了脑组织，脑膜脑。切断大脑的腹侧方颅神经。 </li><li>其余的大脑小脑的分离是很重要的原因有两个。首先，它允许前小叶，这是从视图中隐藏在原位丘表达模式的探索。二是组织分离成更小的，偏远地区，允许为m矿石完全固定，可以方便一些蛋白质染色。纵观这最后的解剖步骤，应注意不要触摸钳提示小脑。插入的优势和劣势丘中间的钳子提示。与第二组的镊子慢慢逗了小脑下丘。然后，奠定了小脑，因此，其前侧朝下和脑干朝上的。插入脑干和小叶第IX /小脑的X之间的第四脑室钳。剪下的梗用钳子捏两侧，然后轻轻抬起小脑脑干。小脑是孤立的，一旦发布修复浸泡在4°C 24-48小时最低为4％煤灰。小脑可以长时间储存​​在4％的煤灰。脑膜应更好地表达模式的知名度染色前或后删除。如果实验者选择删除脑膜染色前有一个强大的可能性，在这个过程中，小脑将缺口。在染色过程结束剥去脑膜要简单得多，因为他们更容易找到后，他们获得了弱的背景染色，后处理成为体弱。 </li></ol> 2。 Wholemount染色的组织处理因为wholemount染色方法需要较长的时间比组织切片的免疫组织化学染色，它是帮助每个实验计划的时间表（ 见表2），例如日历显示。在开始之前，有几个关键的事情要记住。 1）含有的微管组织应该在任何时候都nutator旋转，除在冻结/解冻过程。 2）有几种解决方案必须为每个实验的新鲜（见解决方案食谱表3）。 3）整个协议的，变化的解决方案时，轻轻地倒出来，而不是用钳子取出小脑花了解决方案，以避免接触小脑。然后，在另一个容器中混合后，新的解决方案，用吸管轻轻地添加新的解决管。 2.1第1天： <ol class="lalpha"><li>在室温下固定6-8小时上nutator轻轻摇摆的小脑在登特的修复3。如果甲醇是破坏性的抗原，可以考虑更换步骤，2.1A，2.3A通过与抗原检索方法。抗原检索的热量和使用的缓冲区，将有助于该组织准备抗体渗透。 </li><li>漂白小脑登特的漂白剂3一夜之间在4°C。 </li></ol> 2.2第2天： <ol class="lalpha"><li>在100％甲醇在室温下两轮各30分钟，脱水小脑。 </li><li>然后，以加强渗透的解决方案，组织了一系列冻结/解冻周期。小心地放置在一个容器干冰和30分钟的冻结管。然后，取出试管，并留在室温15分钟替补。冻结/解冻步骤应执行了4次（至少）。 </li><li>组织的最后解冻后，将管到-80°C冰箱过夜。 </li><li>组织可以长时间储存​​在-80°C间立即冻结/解冻步骤之前或之后。否则，该协议应继续下去。 </li></ol> 2.3第3天： <ol class="lalpha"><li>水化PBS洗涤50％小脑MeOH/50％，15％MeOH/85％PBS，100％FBS为60-90分钟，在室温。 </li><li>接下来，成年小鼠小脑，受到2-3分钟10μg/mL组在PBS中的蛋白酶K消化酶组织。没有消化步骤是必要的年轻动物的小脑（P5或鼠标年轻）。不再digestion是提出更大的大脑。例如，可以消化5-6分钟4个灵长类动物的大脑一样高。 </li><li>消化后，立即在PBS洗三次小脑在室温下，每次10分钟，以去除蛋白酶K </li><li>阻止组织中的光电倍增管（ 见表3），4过夜°C。奶粉往往是选择阻断剂对蛋白质的工作时的首选，因为它价格低廉，有效降低背景染色，通常不会干扰抗原抗体结合或访问。动物血清中也可以使用，但实验者应该意识到增加的成本，必须先测试血清交叉反应的免疫球蛋白和能力，以产生最佳的信噪比质量。 </li></ol> 2.4 4-5日： <ol class="lalpha"><li>孵育的组织在光电倍增管稀释的初级抗体辅以48小时在4℃（LARG 5％DMSO呃小脑会需要增加孵育时间）。 </li></ol> 2.5第6天： <ol class="lalpha"><li>小脑洗净，在4°C 2-3次，以除去未结合的抗体，在光电倍增管每2-3个小时。 </li><li>然后，孵化组织在二级抗体，在含5％DMSO的光电倍增管的解决方案4°C过夜（较大的小脑可能需要增加孵育时间）。 </li></ol> 2.6第7天： <ol class="lalpha"><li>小脑洗净的2-3倍，在光电倍增管每2-3个小时，在4°C </li><li>给予组织与PBT（ 见表3）为1-2小时，最后洗净。这一步去除多余的牛奶和提高染色的清晰度。 </li><li>最后，孵化在DAB（3,3&#39; - 二氨基联苯胺）解决方案的组织，直到达到最佳的染色强度。棕色的反应产物，应该是〜10分钟内清晰可见。最好是民建联在黑暗中使用轻造成的发色来降水TE，它可以增加背景染色。 </li></ol> 2.7 当达到最佳的染色强度，停止放置在PBS 0.04％叠氮化钠与小脑的反应。组织可以存储长期在此解决方案。叠氮化钠是一种细菌生长的有效抑制剂，但应避免到这一步，因为它也抑制辣根过氧化物酶。 2.8可选放大过程： 按照正常的协议，但应在地方2.5B-2.7以上的步骤执行这些步骤。 <ol class="lalpha"><li>孵育组织一夜之间在4°，生物素标记抗体，PBST与5％二甲基亚砜（ 见表3） </li><li>冲洗组织PBST 3-4在室温每2-3小时。 </li><li>孵育过夜小脑4°Ç在PBST农行在复杂的解决方案。 </li><li>每2-3小时冲洗组织在3-4变化的PBS室温。 </li><li>孵育新鲜准备民建联解决方案中的组织，如上所述。 </li><li>当染色是最优的，停止由0.04％叠氮化钠的PBS洗涤小脑的反应。 </li></ol> 3。成像的彩色组织<ol><li>可以通过使用实例，抓获wholemount图像，的徕卡DFC3000 FX相机上安装运行徕卡应用套件FX软件徕卡MZ16足总杯体视显微镜。如果需要，卷积显微镜可用于获得不同焦平面多个连续的图像，每个图像只有一个清脆的重点单小叶。然后，图像可以被压缩成一个单一的协议栈和软件渲染消除失真。最后的合成图像，说明小脑表面的表达模式，明确的重点中的所有可见的图案。 </li><li> wholemount染色组织应成像，而沉浸在PBS（或其他媒介，将给予最佳折射率）。为了方便定位的成像wholemount，在塑料培养皿1％琼脂糖凝胶。切成凝胶的小孔。孔将允许小脑楔入到想要的方向。 </li><li> Adobe Photoshop CS4中的原始数据导入和调整对比度和亮度水平。 </li></ol>动物经核准的的IACUC动物协议下进行所有的动物实验研究，根据阿尔伯特·爱因斯坦医学院的体制指引。男性和女性的远交瑞士韦伯斯特（Taconic的，奥尔巴尼，纽约州）小鼠保持在我们的殖民地，并为所有的研究。安乐死的成年大鼠请提供由博士布赖恩约旦（阿尔伯特·爱因斯坦医学院）。所有的动物至少一个月。 4。代表结果小脑分子表达的是条块分割，横向分为四个区域：前区（AZ：〜小叶四），中央区（锆石：小叶六，七），后区（PZ值：〜小叶第八背九）和结节区（新西兰：小叶九腹侧和X ）5。每个区域都包含的矢状条纹（ 图1）1,2,5,6独特的阵列。 Purkinje细胞ZebrinII在表达揭示条纹AZ和PZ（ 图2），CZ和新西兰元（ 图2）统一表达。矢状窦旁组织的Purkinje细胞是反映传入纤维的终端领域地形。可卡因和安非他明调节转录肽（CART）的攀登纤维（ 图3a）的子集表示，该项目在小脑皮层7（ 图3b）的分子层的Purkinje细胞树突的条纹。用适当的修改，如放大7，wholemount协议允许可视化olivocerebellar模式没有的东东一个艰苦的，耗时的重建时间从染色组织切片（ 图3b）。 <img alt="图1" src="/files/ftp_upload/4042/4042fig1.jpg" /> 图1。的表达A. ZebrinII / aldolaseC揭示矢状乐队在小脑的横截面。比例尺= 500微米。B. ZebrinII / aldolaseC表示只在Purkinje细胞胞体和树突。比例尺=150μm的（GL =颗粒层; PCL = Purkinje细胞层;毫升=分子层）。 <img alt="图2" src="/files/ftp_upload/4042/4042fig2.jpg" /> 图2。一个wholemount的小脑沾上为ZebrinII / aldolaseC显示图像前（AZ），中央（CZ），后区（PZ值），小脑的Purkinje细胞的条纹。在这里，我们也表明例如刻痕小脑的消极后果。造成artifactual染色显示红色星号。比例尺= 1毫米（LS = lobulus单纯PML的正中小叶;缔约方会议=系词pyramidis）。 <img alt="图3" src="/files/ftp_upload/4042/4042fig3.jpg" /> 图3表示在登山分子层中的纤维终端A.车。比例尺= 100微米。（毫升=分子层; PCL = Purkinje细胞层; GL =颗粒层）B. Wholemount车在新西兰的表达免疫组化。比例尺= 2毫米（缔约方会议=系词pyramidis;维甲酸=小叶正中; PFL = paraflocculus;佛罗里达=绒球​​）。

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P., Mishina, Y., Rabinovitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SM22alpha-targeted+deletion+of+bone+morphogenetic+protein+receptor+1A+in+mice+impairs+cardiac+and+vascular+development,+and+influences+organogenesis.">SM22alpha-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis.</a> Development. 135, 2981-2991 (2008).</li><li>Mondrinos, M. J., Koutzaki, S., Lelkes, P. I., Finck, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-engineered+model+of+fetal+distal+lung+tissue.">A tissue-engineered model of fetal distal lung tissue.</a> Am. J. Physiol. Lung Cell Mol. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+mapping+of+afferent+topography+in+three+dimensions.">Fluorescence mapping of afferent topography in three dimensions.</a> Brain Struct. Funct. 216, 159-169 (2011).</li><li>Davis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemistry.">Whole-mount immunohistochemistry.</a> Methods Enzymol. 225, 502-516 (1993).</li></ol>

我们什么都没有透露。

我们已经介绍了使用一个多功能的免疫组织化学方法，在发展中国家和成人的大脑暴露蛋白表达为成功wholemount的染色所需的技术细节。复杂的分子的表达方式，通过使用这种方法，可以分析和脑地形而不需要费时费力组织切片程序赞赏。 该协议已被用于揭示成人1,2,8,9和产后早期小鼠小脑10,11,12图案几个Purkinje细胞蛋白表达。我们原来的研究也表明了实用的的?...

<table border="1"><tbody><tr><td> 物料 </td><td> 在协议中的作用 </td></tr><tr><td>灌注泵（费舍尔Scientific/13-876-2） </td><td>允许一致的和缓慢灌注。 </td></tr><tr><td>夏普尖端剪刀（FST/14081-08） </td><td>一般用在灌注和解剖。 </td></tr><tr><td>钝尖钳（FST/91100-12） </td><td>为了稳定灌注针插入心脏。 </td></tr><tr><td>镊子（杜蒙AA/11210-10由福斯特） </td><td>对于使用过程中的脑由颅骨的解剖和大脑的其余部分分开小脑。这些都是必要的，因为他们有一个尖端略圆，有助于最大限度地减少损害小脑在解剖。 </td></tr><tr><td> nutator（Fisher Scientific则） </td><td>用来保持在运动过程中的潜伏期组织。 </td></tr><tr><td> 1.5 mL管（萨尔斯塔特/螺帽微管） </td><td>在他的所有步骤组化协议，在这些微管的地方。圆润的底部，确保小脑在运动中保持。 </td></tr><tr><td>穿孔勺（FST/10370-17） </td><td>用于轻轻滗出花的解决方案保持在微管wholemounts。 </td></tr><tr><td>徕卡MZ16足总杯显微镜</td><td>用于检查wholemount染色。 </td></tr><tr><td>的徕卡DFC3000 FX相机</td><td>用来捕捉wholemount图像。 </td></tr></tbody></table> 表1。 <table border="1"><tbody><tr><td colspan="9"> 例如日历典型wholemount实验 </td></tr><tr><td>第1天</td><td colspan="4">登特的修复，室温，8小时</td><td colspan="4">登特的漂白剂，4℃，过夜</td></tr><tr><td>第2天</td><td colspan="3"> 100％甲醇，室温，2X，每次30分钟</td><td colspan="3"> 100％甲醇，冻结/解冻， 4倍，30 min/15分钟</td><td colspa<html> N =“2”&gt; 100％甲醇，80°，隔夜</td></tr><tr><td>第3天</td><td> 50％MeOH/50％PBS，室温60-90分钟</td><td colspan="2"> 15％甲醇/ 85％PBS，室温60-90分钟</td><td colspan="2"> 100％PBS，室内温度60-90分钟</td><td> 10μg/mL组蛋白酶K在PBS，室温2-3分钟</td><td> 100％FBS，室温，3倍，每10分钟</td><td>光电倍增管，4℃，过夜</td></tr><tr><td> 4-5天</td><td colspan="8">光电倍增管+ 1°抗体+ 5％DMSO，4°C间，48小时</td></tr><tr><td>第6天</td><td colspan="3">光电倍增管，4℃，2 - 3倍，每2-3小时</td><td colspan="5">光电倍增管+ 2°抗体+ 5％DMSO，4°C间，24小时（或开始与ABC复杂扩增步骤） </td></tr><tr><td> 7天</td><td colspan="2">光电倍增管，4℃，2 - 3倍，每2-3小时</td><td colspan="3"> PBT，室温，2小时; </td><td colspan="3">新鲜民建联在PBS孵育，直到最佳染色是可视化</td></tr></tbody></table>_content“&gt; 表2。 <table border="1"><tbody><tr><td colspan="2"> 食谱 （* =每次都准备好新鲜） </td></tr><tr><td> PBS（磷酸盐缓冲液） </td><td> 0.1M磷酸盐缓冲液中去离子水。 pH值7.2（西格玛片; P4417） </td></tr><tr><td> PFA（聚甲醛） </td><td>制造和储存冻结20％的解决方案，然后4％的PBS稀释为工作液（Fisher Scientific则; T353） </td></tr><tr><td>登特的定影液3 * </td><td> 4部分甲醇 第1部分二甲基亚砜（二甲基亚砜; Fisher Scientific则; D159-4） </td></tr><tr><td>登特的漂白剂3 * </td><td> 4部分甲醇 第1部分二甲基亚砜（二甲基亚砜; Fisher Scientific则; D159-4） 第1部分30％的过氧化氢</td></tr><tr><td>酶解</td><td> 10微克/毫升的PBS，蛋白酶K（罗氏诊断; 03115828001）。 </td></tr><tr><td> PBST </td><td> PBS包含： 0.1％的吐温20（Fisheŕ科学，BP337;海卫也可以在所有情况下使用Tween-20的地方）。 </td></tr><tr><td>光电倍增管25 * </td><td> PBS包含： 2％脱脂脱脂奶粉（康乃馨优先） 0.1％Tween-20的（Fisher Scientific则BP337） </td></tr><tr><td> PBT的25 * </td><td> PBS包含： 0.2％的牛血清白蛋白（Sigma公司; B9001S） 0.1％Tween-20的（Fisher Scientific则BP337） </td></tr><tr><td>民建联* </td><td>溶解在40毫升的PBS 3,3 - 二氨基联苯胺10毫克片剂（Sigma-Aldrich公司; D5905）。加入10μl30％的过氧化氢的启动反应）。 </td></tr><tr><td> ABC复杂的解决方案</td><td> vectastain套件（媒介实验室，公司PK-4000） </td></tr></tbody></table> 表3。

wholemount immunohistochemistry for revealing complex brain topography

反复和充分了解小脑的细胞结构，使其理想的模型系统，为探索脑地形。其相对统一的细胞结构的基础是一系列复杂的基因和蛋白表达的矢状域。小脑分子条块分割镜像传入纤维的解剖和功能的组织。要充分认识小脑组织的复杂性，我们以前完善了wholemount染色方法在小鼠小脑的图案缺陷的高通量分析。该协议详细描述的试剂，工具，是有用的实际步骤，成功地揭示了成年小鼠小脑使用wholemount染色蛋白表达模式。这里展示的步骤强调使用这种方法的效用作为一个例子，如何可以在其透露的大脑的精细地形表达zebrinII / aldolaseC的原生立体构象。还介绍了协议允许传入的预测和大量分子地形比较研究小脑蛋白表达的可视化的适应。为了说明这些应用程序，从大鼠小脑的传入染色的数据。

Watch this Scientific Journal Video about Wholemount Immunohistochemistry for Revealing Complex Brain Topography at JoVE.com

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

神经回路地形组织功能的车厢与特定的分子型材。在这里，我们提供一个多功能wholemount免疫组织化学染色方法为揭示全球脑地形使用的实际和技术步骤。我们证明了实用的使用方法很好理解的小脑细胞构筑和电路。

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

wholemount-immunohistochemistry-for-revealing-complex-brain-topography

Research

JoVE Journal

Neuroscience

15.5K 次观看.  Albert Einstein College of Medicine, Yeshiva University. 神经回路地形组织功能的车厢与特定的分子型材。在这里，我们提供一个多功能wholemount免疫组织化学染色方法为揭示全球脑地形使用的实际和技术步骤。我们证明了实用的使用方法很好理解的小脑细胞构筑和电路。

视频: Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an extensively studied model system for understanding the behavior of neural stem cells and the regulation of adult neurogenesis. Traditionally, these studies have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region. Compared to sections, wholemounts preserve the complete cytoarchitecture and cellular relationships within the SVZ. This approach has recently revealed that the adult neural stem cells, or type B1 cells, are part of a mixed neuroepithelium with differentiated ependymal cells lining the lateral ventricles. In addition, this approach has been used to study the planar polarization of ependymal cells and the cerebrospinal fluid flow they generate in the ventricle. With recent evidence that adult neural stem cells are a heterogeneous population that is regionally specified, the wholemount approach will likely be an essential tool for understanding the organization and parcellation of this stem cell niche.

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain. Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis. Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal region.

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

科研

神经科学

Revealing Neural Circuit Topography in Multi-Color

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably label the global patterning of multiple topographic projections. The cerebellum is an ideal model to study the orderly arrangement of neural circuits. For example, the compartmental organization of spinocerebellar mossy fibers has proven to be an indispensable system for studying mossy fiber patterning. We recently showed that wheat germ agglutinin (WGA) conjugated to Alexa 555 and 488 can be used for tracing spinocerebellar mossy fiber projections in developing and adult mice (Reeber et al. 2011). We found three major properties that make the WGA-Alexa tracers desirable tools for labeling neural projections. First, Alexa fluorophores are intense and their brightness allows for wholemount imaging directly after tracing. Second, WGA-Alexa tracers label the entire trajectory of developing and adult neural projections. Third, WGA-Alexa tracers are rapidly transported in both retrograde and anterograde directions. Here, we describe in detail how to prepare the tracers and other required tools, how to perform the surgery for spinocerebellar tracing and how best to image traced projections in three dimensions. In summary, we provide a step-by-step tracing protocol that will be useful for deciphering the organization and connectivity of functional maps not only in the cerebellum but also in the cortex, brainstem, and spinal cord.

We provide a practical guide for delivering tracers in vivo and use the spinocerebellar pathway as a model system to demonstrate essential steps for successful neuronal circuit analysis in mice. We describe in detail our versatile tracing protocol that exploits wheat germ agglutinin (WGA) conjugated to Alexa fluorophores.

多色揭示神经回路地形

Neural circuits are organized into functional topographic maps. In order to visualize complex circuit architecture we developed an approach to reliably ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织：例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

常见马莫塞的全皮质电皮质阵列的慢性植入

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...