共聚焦图像，获得爱荷华大学卡弗中心成像。 NIDCD（5R01DC00559007）提供资金SBIR资助（MH079805）提供额外的支持这个项目的小鼠繁殖的BF的BF

1。亲油性染料注入组织准备： 所有的组织解剖和操作进行了动物在4％多聚甲醛（PFA）固定在0.1 M磷酸缓冲液（pH值7.4）transcardial使用适当大小的针头蠕动泵的灌注。组织可存放在冰箱长达6个月在4％煤灰。所有的准备工作和操作都在0.4％或更高的煤灰进行，直到安装甘油成像。这煤灰量，有效地消除了核糖核酸酶的RNA，并保留在原位杂交。 染料存储： 所有染料应保存在一个黑暗的封闭车厢，以减少空气流通和接触到明亮的日光，因为他们是感光。为了避免交叉污染，应单独设置的仪器用于处理每个染料。 <ol><li>首先，必须选择和染料的应用程序网站无关的组织中提取，以直观地确认所选择的网站，染料的位置访问。选择申请地点是在这个过程中最重要的一步。选择标签正在审议的神经元群的一个很好的网站，并没有多余的结构可以是具有挑战性。安置成道在解剖范围的可视化控制下的精度要求的某些问题的神经解剖学的认识水平。染料可以放在中央或外围标签对于一个给定的项目（即脑，周围神经，耳，眼），分别为所需的外围或中央的预测。 Lipophic染料将弥漫在所有属于一个给定的神经元的过程，逆行和anterogradely，填充一个给定的神经元或所有的神经元投射到一个给定的轨道。 </li><li> NeuroVue MTTI染料预先加载到应用简便的滤纸条。染料可以溶解于100％二甲基亚砜（工作引擎盖下）和稀疏的头发，可以浸泡在这种解决方案，随后干较小的应用程序。预装的滤纸染料切成适当大小的三角块，与microscissors。染料的大小将取决于大小的标本，被标记的结构大小，并同时使用的颜色数。请务必尽可能削减了一块小标签等结构，以避免。在使用切割和处理染料搅拌，在酒精的仪器，以消除任何残留染料，然后完全风干，或用纸张民建联钳microsissors。这是为了避免标本污染文书标签不必要的结构与残余染料。 </li><li>对于插入到软组织，如大脑的过滤器可直接插入使用过滤器的三角点，皮尔斯组织。对于更严格的组织作出的切口，以便更容易插入染料。不要用解剖针推入组织的过滤器。这可能会导致不准确的位置，组织和混乱。插入备用NeuroVue染料波长作为额外的神经元群体的标签需要。 </li><li>插入的染料，并核实其位置后，标本放置在一个安全封闭的小瓶，用4％的煤灰和培养36 ° C黑暗环境中为2-14天，根据年龄和扩散距离被覆盖（约2毫米，每天36℃）。 </li><li>可以使用一个与epiflouresence解剖范围来评估，如果染料扩散到所需位置，标本可放置在孵化器的扩散被认为是不够。使用没有epifluorescene一个正常的解剖检测扩散的范围会低估真实长度的染料已经扩散。另外，如果染料浓度足够高，以评估视觉上可能会导致吸收淬火时发出荧光。 </li><li>解剖出所需的组织利益和整个安装到与甘油的幻灯片和共聚焦显微镜成像coverslipped。共聚焦设置为1，2中所述。如果组织规模的抑制整个幻灯片上安装，需要连续切片（见下文）。组织准备成像和染料的光谱特性的更多细节可在<a href="http://www.mtarget.com/mtti/documents/NeuroVuebrochuremar2010.pdf">：</a> http://www.mtarget.com/mtti/documents/NeuroVuebrochuremar2010.pdf </li></ol><img src="/files/ftp_upload/2451/2451fig1.jpg" alt="图1" /> 图1。实验的示意图。耳暴露，让所有结构的可视化。亲油性染料，然后注入允许弥漫。后扩散，由不同的染料标记，可以看出不同的神经元群体。接下来， 在原位杂交（Sox2基因）在耳朵上和标签的mRNA表达。然后进行了免疫组织化学（Myo7，微管蛋白），可视化蛋白的表达。其次是组织切片原位杂交和免疫组织化学可以可视化。 在原位杂交2。 如果你想开始原位杂交（后与亲油性的染料跟踪将是不可能的），请参阅第1部分中的组织准备。每次洗，除非另有说明，进行了在室温下对逆变器/混频器与2毫升的溶液。一定要在一个干净，核糖核酸酶自由的环境中工作。 <ol><li>脱水，然后通过一个分级的甲醇系列补充水分的样品。 <ol class="lalpha"><li> 100％1小时通宵@ 4 ° C（可选：样品在100％的甲醇储存在-20 ° C） </li><li> 75％× 5分钟。 @ 4 ° C </li><li> 50％× 5分钟。 @ 4 ° C </li><li> 25％× 15分钟。 @ 4 ° C（或直至组织汇） </li><li>样品转移到2 mL Eppendorf管（核糖核酸免费）。 </li></ol></li><li>在PBS（1X PBS）洗三次，并打开以供将来使用（60℃）杂交炉。 <ol class="lalpha"><li> PBS × 5分钟。 </li><li> PBS × 5分钟。 </li><li> PBS × 5分钟。 </li></ol></li><li>精华2μL20毫克/毫升的股票蛋白酶K（Ambion公司猫＃AM2546）2.0 mL新鲜PBS组织。 实际PK消化时间取决于胚胎的年龄。下面是一个时代的粗略估计。消化，应密切监测deproteination是关键的一步。在消化会导致贫困探针的渗透，而结构完整性的丧失将导致过度消化。一个良好的消化指标是在该组织从不透明的变化几乎清除。 <table border="1"><tbody><tr><td colspan="2">表1。对小鼠组织蛋白酶K的消化时间。 </td></tr><tr><td>年龄（胚胎一天） </td><td>时间（分钟） </td></tr><tr><td> E8.5 </td><td> 6 </td></tr><tr><td> E9.5 </td><td> 10 </td></tr><tr><td> E10.5 </td><td> 13 </td></tr><tr><td> E11.5 </td><td> 15 </td></tr><tr><td> E12.5 </td><td> 17 </td></tr><tr><td> E14.5 </td><td> 18 </td></tr><tr><td> E16.5 + </td><td> 20 +的</td></tr></tbody></table></li><li>停止孵化5分钟的样品在4％煤灰的消化。 </li><li>洗的PBS。 <ol class="lalpha"><li> PBS × 1分钟。 </li><li> PBS × 5分钟。 </li><li> PBS × 5分钟。 </li><li> PBS × 5分钟。 </li></ol></li><li>倒掉PBS特别注意尽可能消除。 </li><li> Prehybridize样本：孵育在60 °的C（1.8毫升至少1小时的杂交组合中的变频器）。设置IsoTemp热块到85 ° C以供将来使用。 <ol class="lalpha"><li>由于一小时的临近，变性鲑鱼精子DNA（Invitrogen公司CAT号15632-011）孵育在85 ° C为10分钟。设置上，直到用冰。 </li></ol></li><li>预杂交至少1小时后，加200μL的变性ssDNA和每个样品约100ng高辛标记的riboprobe。在60℃杂交炉杂交过夜。 </li><li>更换2毫升2X SSC杂交组合。 IsoTemp热块设置至37 ° C和70 ° C，以供将来使用。 <ol class="lalpha"><li>与2X SSC × 10分钟清洗。 @ 60℃杂交炉。 </li><li>与2X SSC × 10分钟清洗。 @ 60℃杂交炉。 </li><li>与2X SSC × 10分钟清洗。 @ 60℃杂交炉。 </li><li>与2X SSC × 60分钟清洗。 @ 70 °彗星在IsoTemp加热块。 </li></ol> 70 ° C时删除任何内源性碱性磷酸酶活性。这也许是最重要的一步，对降低背景。第（四）可能被分成两个30分钟。清洗，以尽量减少对组织的损害。 </li><li>洗涤用PBS × 5分钟。解冻核糖核酸酶上以供将来使用的冰。 </li><li>更换PBS和添加1.0μLRNA酶A酶（Fermentas公司EN0531 10毫克/毫升）× 60分钟。 @ 37 °彗星IsoTemp加热块（90分钟。潜伏期是首选，如果是高度集中的探头）。 </li><li>丢弃PBS / RNA酶A和洗4次。 <ol class="lalpha"><li> 1X洗涤液× 10分钟。 </li><li> 1X洗涤液× 10分钟。 </li><li> 1X洗涤液× 10分钟。 </li><li> 1X洗涤液× 60分钟。 @ 70 °彗星在IsoTemp加热块。 </li></ol></li><li>在1X块缓冲区孵育1小时。 <ol class="lalpha"><li>此时，准备2.0毫升和1μL，抗地高辛抗体（1:2000），每个样品块缓冲区。简要反转，让坐在4 °，直到使用C。 </li></ol></li><li> 1小时后丢弃块缓冲区和样品添加2.0毫升吸收块缓冲区+抗体。 </li><li>孵育过夜。 </li><li>丢弃块解决方案。 </li><li>与1X缓冲液洗清洗。 <ol class="lalpha"><li> 1X缓冲液洗× 5分钟。 </li><li> 1X缓冲液洗× 5分钟。 </li><li> 1X缓冲液洗1小时x 5-6变化。 </li></ol></li><li> 1X缓冲液洗，洗过夜。 </li><li>冲洗1X detection缓冲10分钟。 </li><li>将样品检测缓冲区孔板。 </li><li>删除缓冲区，并与BM紫色（罗氏公司11442074001）检测，直到获得所需的信号强度（较长的探头，这可能意味着在一夜之间）。兽王紫色光敏感盖用铝箔或一箱。 <ul><li>使用前混合骨髓紫。 </li><li>确保样品自由浮动，而不是坚持井。 </li><li> 1小时后，检查信号。 </li></ul></li><li> 1X井× 5分钟检测缓冲液冲洗样本。 </li><li>图像或存储样品在4％PFA在4 ° C </li></ol> *样品可以在这个阶段成像和/或免疫组化步骤后添加（下文第3部分）。 解决方案： <ul><li>核酸游离水：1L无菌超纯水的混合残留的DEPC（Sigma公司）1毫升。高压灭菌。 </li><li> 1X PBS：将1 PBS包（Sigma公司）在1L核酸自由水和过滤消毒。 </li><li> 1X洗涤液混合核酸游离水450毫升+ 50毫升10倍洗涤液和过滤消毒。 </li><li>杂交组合：50％的体积（25毫升）甲酰胺; 50％2X SSC体积（25毫升）;按重量的6％右旋糖酐硫酸（3G）。 </li><li> 1X检测缓冲液：混合50毫升与450毫升核酸免费的水和过滤器的10倍检测缓冲液消毒。 </li><li> 1X块缓冲区：5毫升10X马来酸缓冲液（罗氏缓冲区设置），40毫升核酸自由水; 5毫升10X块缓冲区。 </li><li> 2X SSC：用450毫升核酸免费的水和过滤器的混合50毫升20X SSC的消毒。 </li><li>分级甲醇：核酸至75％的自由水，50％，25％的浓度稀释100％的甲醇。 </li></ul> 3。免疫组织化学可以省略步骤1和2，如果第2部分完成。在这种情况下，确保所有甘油或其他安装介质是洗掉。请注意，并非所有的抗体仍然能够检测蛋白酶K消化后的抗原表位。我们有一个短名单，可用于在原位杂交技术在哺乳动物组织在保留其特殊性，因为他们（见表2）结合的抗体。 <ol><li>梯度乙醇系列，摆脱亲脂性染料的组织（如果没有完成效果在第2部分）：50％乙醇5分钟，70％乙醇5分钟，95-100％的乙醇过夜或根据需要，直至达到预期效果，70％的乙醇5分，50％的乙醇5分钟。 </li><li>通过逐步增加PBS在5-10分钟内补充水分。 </li><li>座为1小时，2.5％正常山羊血清（NGS）和0.5％Triton X - 100的的室温摇床组织。 （1:1 5％的农工商超市：1.0％TritonX - 100 1X PBS） </li><li>孵育与初级抗体稀释块解决方案48-72小时@ 4 ° C的摇床（S）。 </li><li>用PBS洗1小时× 3的变化。 </li><li>在步骤3座。 </li><li>孵育块解决方案的二次抗体稀释为12 - 24小时（S）@ 4℃摇床（使用的Alexa Fluor ®染料自Invitrogen）。 </li><li>在第5步洗。 （股权。计数器用Hoechst核染色染色先洗稀释1小时10毫克/毫升的PBS股票1:2000洗PBS x 2-3变化：上筛RT） </li><li>影像学检查：标本可以在这一步，传输和啶显微镜成像，优先使用共焦成像系统的一个补充决议。要做到这一点，我们经常在甘油中的整个安装耳，用盖玻片作为垫片，以避免过度压缩的组织。此外，而不是这个整装成像或标本，可以嵌入在环氧树脂连续切片病理细节补充。要受益于合并后的整个挂载/节的分析，避免漂白的荧光信号中的共焦成像步骤。 </li></ol>解决方案<ul><li>乙醇溶液：蒸馏水稀释100％乙醇至终浓度为50％和70％。 </li><li>座解决方案：1:1稀释5％农工商超市：1.0％TritonX - 100 1X PBS（最后工作浓度：2.5％农工商超市; 0.5％TritonX - 100）。 </li><li> 1X PBS：将1 PBS包（Sigma公司）在1L蒸馏水。 </li><li> 5％的农工商超市：解冻2.5毫升农工商超市和47.5 mL 1X PBS中混合。保存在4 ° C。 </li><li> 0.5毫升1.0％TritonX - 100和49.5 mL的1X PBS，TritonX - 100（离开摇床@ RT混合1小时）。保存在4 ° C。 </li><li>赫斯特染色储备溶液：溶解10毫克每1X PBS毫升Hoechst染料。 </li></ul><table border="1"><tbody><tr><td colspan="3">表2。抗体，蛋白酶K消化组织的工作。 </td></tr><tr><td> 猫＃ </td><td> 项目 </td><td> 协力厂商 </td></tr><tr><td> 25-6790 </td><td>抗肌球蛋白VIIA部多克隆</td><td>变形杆菌生物科学</td></tr><tr><td> 25-6791 </td><td>抗肌球蛋白VI多克隆</td><td>变形杆菌生物科学</td></tr><tr><td> 9661 </td><td>劈开CASPASE - 3多克隆</td><td>细胞信号转导</td></tr><tr><td> T7451 </td><td>微管蛋白的单克隆抗-乙酰人</td><td>西格玛</td></tr><tr><td> PRB - 238C </td><td> Prox1多克隆</td><td> Covance公司</td></tr></tbody></table> 4。组织学切片后可以推出第1部分，以增加亲脂厚如少年的大脑或2或第3部分完成后整个坐骑跟踪染料决议。对于串行的脑切片中，我们建议Compresstome VF - 700切片机（Precisionary仪器公司，格林维尔，北卡罗来纳州），获得均匀的部分厚度。冰冻切片优化的较少，由于在切片过程3中的更大的染料渗漏。连续切片的制备和安装在甘油 4是组织编制的首选方法，整个支架或表面安装时不允许足够的可视化组织地区利益。组织也可以随后被嵌入在环氧树脂中，并减少使用透射电子显微镜的玻璃或钻石刀在1-2微米厚度。当使用这种技术最免疫仍将是塑料包埋后更加稳定，但是，所有的亲脂性的跟踪将完全失去了在这个阶段，因为它们溶解在酒精和环氧树脂。采取预防措施作为样本将光敏感，直到它们被嵌入。 Compresstome VF - 700切片机切片，按照Precisionary仪器公司的建议环氧嵌入/分段： <ol><li>固定：gluteraldehyde 2.5％/ 1％PFA在0.1M磷酸盐缓冲液（pH值7.4），1小时至过夜。 </li><li>在一个玻璃样品瓶脱水组织：30％乙醇（5min.），50％的乙醇（5min.），70％乙醇（5min.隔夜），95％的乙醇（5倍的10分钟。），无水乙醇（5X 10分钟每件）。 </li><li>过渡溶剂：1:1的无水乙醇：环氧丙烷（PO）的5分钟。 </li><li>溶剂：订单仅5倍的10分钟。每个。 </li><li>渗透与树脂： <ol class="lalpha"><li> 1:1 PO：树脂摇床过夜。 </li><li>倒入平嵌入模具与样品溶液（S），并离开柜台4-6小时蒸发宝。另外，去掉瓶盖，摇床离开4小时（与更大的组织）。 </li><li>传送样本，以100％的树脂为4小时。 </li></ol></li><li>嵌入在标签的最终模具的树脂。聚合，通过在60℃的孵化，为24-48小时。 </li><li>用刀片剪切块需要，尽量减少多余的树脂。切1-2微米Ultramicrotome（Ultracut发送赖克特荣）连续切片。安装和使用啶显微镜图像。 可选：染色与组织的污渍节的一个部分（即Stevenel蓝色）如果需要的话（实现免疫不会持续在这些路段）。 </li></ol>解决方案<ul><li>乙醇溶液：在蒸馏水稀释100％的乙醇30％，50％，70％和95％的浓度。 </li><li>固定的解决方案：混合2.5 mL 50％的Gluteraldehyde，5毫升10％的多聚甲醛，和42.5毫升0.1M磷酸盐缓冲液pH值7.4（终浓度：2.5％gluteraldehyde;煤灰4％）。保存在4 ° C。 </li><li>环氧树脂：根据保利/床812嵌入Media/DMP-30套件（Polysciences公司＃08792-1）附带的使用说明书。储存在-20 ° C。 </li></ul> 5。代表性的成果<img src="/files/ftp_upload/2451/2451fig2.jpg" alt="图2" /> 图2。在小鼠胚胎中的亲脂性染料安置和成像三种不同波长的亲脂性染料注入和培养，让三叉神经（TN）的，舌咽神经（GN），和迷走神经（VN）的中央预测的可视化。一）分为三组颅神经的外围部分亲脂性染料的位置，让扩散到脑干中央流程可视化。 TN是标有红色NeuroVue，GN：NeurVue栗色，和VN：NeuroVue玉。二）（一）孵化后的相同的鼠标。明显微镜，可以看到一些扩散。 c）在相同的鼠标（A和B）。后脑已被解剖和平面安装。一些中央的三个标记神经过程标签可以看出，随着明显微镜。四）（c）的共聚焦图像。共聚焦特定神经元可以看出，和使用三个不同的染料，允许每个人口分布在其他的评估。染料成像顺序。 NeuroVue玉为488 nm的激发和发射500-550纳米成像。 NeuroVue红，在535 nm，发射激发550-600纳米成像。在643 nm和排放量在650-700 nm的激发成像NeuroVue栗色。 <img src="/files/ftp_upload/2451/2451fig3.jpg" alt="图3" /> 图3实验的示意图。耳朵是暴露，让所有结构的可视化。亲脂性染料然后注射和允许弥漫。后扩散，由不同的染料标记，可以看出不同的神经元群体。接下来， 在原位杂交（SOX2）在耳朵上和标签的mRNA表达。免疫组化，然后进行（Myo7，微管蛋白）的可视化蛋白的表达。其次是组织切片原位杂交和免疫组织化学可以可视化。

<ol>
	<li>Jensen-Smith, H., Gray, B., Muirhead, K., Ohlsson-Wilhelm, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-distance+three-color+neuronal+tracing+in+fixed+tissue+using+NeuroVue+dyes.">Long-distance three-color neuronal tracing in fixed tissue using NeuroVue dyes.</a> Immunol Invest. 36, 763-789 (2007).</li><li>Tonniges, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-+and+bio-physical+characterization+of+novel+violet+and+near-infrared+lipophilic+fluorophores+for+neuronal+tracing.">Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing.</a> Journal of Microscopy. , (2010).</li><li>Bartheld, C. S. v. o. n., Cunningham, D. E., Rubel, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+tracing+with+DiI:+decalcification,+cryosectioning,+and+photoconversion+for+light+and+electron+microscopic+analysis.">Neuronal tracing with DiI: decalcification, cryosectioning, and photoconversion for light and electron microscopic analysis.</a> J Histochem Cytochem. 38, 725-733 (1990).</li><li>Gurung, B., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+embryonic+midbrain+thalamic+and+thalamic+auditory+connection+development+in+mice+as+revealed+by+carbocyannine+dye+injection.">Time course of embryonic midbrain thalamic and thalamic auditory connection development in mice as revealed by carbocyannine dye injection.</a> J Comp Neurol. 479, 309-327 (2004).</li><li>Fritzsch, B., Nichols, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiI+reveals+a+prenatal+arrival+of+efferents+at+the+differentiating+otocyst+of+mice.">DiI reveals a prenatal arrival of efferents at the differentiating otocyst of mice.</a> Hear Res. 65, 51-60 (1993).</li><li>Jahan, I., Kersigo, J., Pan, N., Fritzsch, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurod1+suppresses+hair+cell+differentiation+in+ear+ganglia+and+regulates+hair+cell+subtype+development+in+the+cochlea.">Neurod1 suppresses hair cell differentiation in ear ganglia and regulates hair cell subtype development in the cochlea.</a> PloS ONE. 5, e11661-e11661 (2010).</li></ol>

小鼠组织中收集了大学爱荷华机构动物护理和使用委员会（ACURF 0804066）规定的准则和法规的规定。

在这段视频中，我们展示了四项技术结合起来，以最大限度地通过相关的数据在给定的动物（图3）数据收集和凝聚力的一种方法。这种做法将减少养殖量，因此所需要的时间获得发布的数据，同时提高合作本地化和相关的突变体的影响的信息。虽然利用在这段视频中，成年鼠以及其他动物，如鸡，爪蟾和斑马鱼的胚胎小鼠，以及使用这些技术，可以分析。当使用其他物种的蛋白酶K消化可能需要修改。在这里，我们用不同的荧光NeuroVue染料专门标签3个神经元的利息人口。这些染料的好处是，他们可以在突变小鼠，这可能是问题时，单纯依靠免疫组织化学突变进行分析，可能或可能不会影响使用，而且他们的标签神经元逆行和anterogradely 5 。最近的一项研究也可以同时向六个2标记神经元的人口数量增加。之后，亲脂性染料，跟踪同一组织的利益，就可以进行分析，原位杂交，分析基因的转录，随后可以使用蛋白质分布的免疫组织化学分析。后者的分析，可以辅以详细的组织学，可以添加到表型特征 6 。这多因素分析，有可能获得新的见解相同的动物，否则是不可能的，或者至少需要在更广泛的协议和鼠标育种的范围内通过相关性分析。

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>in situ Hybridization:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PC DIG wash + block buffer set</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1585762</td>
<td>&nbsp;</td>
<tr>
<td>Premix 20x SSC Buffer</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>1666681</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K 20mg/ml</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM2546</td>
<td>&nbsp;</td>
<tr>
<td>Diethyl Pyrocorbonate (DEPC) </td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D43060</td>
<td>&nbsp;</td>
<tr>
<td>Formamide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9037</td>
<td>&nbsp;</td>
<tr>
<td>Dextran Sulfate</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>D20020</td>
<td>&nbsp;</td>
<tr>
<td>BM Purple </td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11442074001</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Dig-AP Fab fragments</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11093274910</td>
<td>&nbsp;</td>
<tr>
<td>RNase A enzyme 10mg/ml</td>
<td>&nbsp;</td>
<td>Fermentas</td>
<td>EN0531</td>
<td>&nbsp;</td>
<tr>
<td>RNase Away</td>
<td>&nbsp;</td>
<td>RPI</td>
<td>7003</td>
<td>&nbsp;</td>
<tr>
<td>Salmon Sperm DNA 10mg/ml</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15632-011</td>
<td>&nbsp;</td>
<tr>
<td>Stericup Filter Unit 0.22&mu;m; 500ml</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SCGVU05RE</td>
<td>&nbsp;</td>
<tr>
<td>Filter Discs 0.2&mu;m; 25mm</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>6780-2502</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate buffered saline PH 7.4 packets</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Immunohistochemistry:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Goat Serum</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G9023</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8532</td>
<td>&nbsp;</td>
<tr>
<td>Hoechst Dye</td>
<td>&nbsp;</td>
<td>Polysciences Inc.</td>
<td>9460</td>
<td>&nbsp;</td>
<tr>
<td>Histology:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Poly/Bed 812 Embedding Media/DMP-30 Kit </td>
<td>&nbsp;</td>
<td>Polysciences Inc. </td>
<td>08792-1</td>
<td>&nbsp;</td>
<tr>
<td>Propylene oxide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>110205</td>
<td>&nbsp;</td>
<tr>
<td>Gluteraldehyde Solution 50%</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151</td>
<td>&nbsp;</td>
<tr>
<td>DPX Mountant</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>44581</td>
<td>&nbsp;</td>
<tr>
<td>Rocker</td>
<td>&nbsp;</td>
<td>MidSci</td>
<td>55D1114</td>
<td>&nbsp;</td>
<tr>
<td>Heat Block</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-715-125D</td>
<td>&nbsp;</td>
<tr>
<td>Rotator</td>
<td>&nbsp;</td>
<td>Thermo</td>
<td>400110Q</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 60&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-625D</td>
<td>&nbsp;</td>
<tr>
<td>Dye tracing:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscissors</td>
<td>&nbsp;</td>
<td>Geuder</td>
<td>G-19775</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps</td>
<td>&nbsp;</td>
<td>E.M.S</td>
<td>72680-F</td>
<td>&nbsp;</td>
<tr>
<td>NeuroVue dye: Maroon, Red, Jade</td>
<td>&nbsp;</td>
<td>MTTI</td>
<td>FS-100(1,2,6)</td>
<td>&nbsp;</td>
<tr>
<td>1X phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-5368</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting scope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>M205 FA</td>
<td>&nbsp;</td>
<tr>
<td>Incubator set to 36&deg;</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>11-690-506DQ</td>
<td>&nbsp;</td>
<tr>
<td>Confocal Microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>LSM 510</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00240</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>&nbsp;</td>
<td>Surgipath</td>
<td>00106</td>
<td>&nbsp;</td>
<tr>
<td>RPI</td>
<td>&nbsp;</td>
<td>Glycerol</td>
<td>G22025-0.5</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (4% and 0.4%)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>T353-500</td>
<td>&nbsp;</td>		</tbody>
		</table>
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combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology

超越单个基因的功能切入基因调控网络的更深层次，需要在一个单一的动物相结合的多种突变。两个或两个以上的基因分析，需要加以补充，无论是在整个安装发展的细胞和组织中的表达改变的详细解决部分机关或与其他基因，其蛋白质的免疫组织化学原位杂交。结合多个基因变异，需要使用CRE或flipase有条件删除的基因和避免胚胎杀伤力。所需的育种计划大幅提升的精力和成本突变基因的数量成正比，与极少数的动物所需的基因修饰的剧目结果，。摊销大量的精力和时间，以获得这几年的珍贵标本携带多个突变，需要组织优化。此外，调查与多种技术的单一动物，使得它更容易与表达谱相关基因缺失的缺陷。我们已经开发出一种技术，以获得一个给定的动物的更透彻的分析;分析同一试样在整个安装机关和部分从几个不同的组织学识别的结构，以及基因和蛋白表达的能力。虽然利用老鼠已经证明了这种技术的有效性，它可应用于广泛的动物。要做到这一点， 我们在原位杂交，免疫组织化学和组织学相结合的亲脂性染料跟踪，整个装载提取最大可能的数据量。

Watch this Scientific Journal Video about Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology at JoVE.com

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

不同的技术组合，以最大限度地从小鼠组织中的数据收集。

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

combining-lipophilic-dye-situ-hybridization-immunohistochemistry

Research

JoVE Journal

Neuroscience

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

25.8K 次观看.  University of Iowa. 不同的技术组合，以最大限度地从小鼠组织中的数据收集。

视频: Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学：

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

科研

神经科学

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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 ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

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 ...