The University Paris 13 supported this work. ASPT was supported by a University Paris 13 fellowship and the "Association Fran&#231;aise pour le Syndrome d'Ondine". FJ was supported by a Laboratory of Excellence GR-Ex fellowship. The GR-Ex (ref ANR-11-LABX-0051) is funded by the program "Investissement d'avenir" of the French National Research agency (ref ANR-11-IDEX-0005-02).

注:C-FOS检测是涉及几个步骤（ 图1）的标准化过程。所有实验在大鼠或小鼠中进行。实验方案由伦理委员会在动物实验查尔斯·达尔文（CE5 / 2011/05）批准，按照2010年9月22日（2010/63 / EU）动物保健，欧洲共同体理事会指令完成，按照实施与法国的法律对动物的照顾。 1.溶液的制备<ol><li>制备磷酸盐的0.2M磷酸钠缓冲液:补充6.24克的NaH 2 PO 4 * 2H 2 O和22.56克Na 2 HPO 4 * H 2 O至1升蒸馏水，同时搅拌。 </li><li>准备4％多聚甲醛（PFA）:添加20克PFA的蒸馏水150毫升加热该溶液至80℃，同时搅拌。要清除溶液，减少热量并添加1N的NaOH的1或2滴，以帮助多聚甲醛溶解。完成至250毫升用蒸馏水和添加250毫升0.2M磷酸缓冲液（pH 7.4）。保存在4℃。使用这些试剂时，采取适当的照顾。带着手套，实验室外套，并且在化学罩护目镜处理。 注:在灌注前一天，准备4％PFA。确定4％PFA的体积的年龄，数目和动物物种的功能。 </li><li>制备冷冻保护溶液:在250ml的0.2M磷酸盐缓冲盐水中，混合5克聚乙烯吡咯烷酮，150克蔗糖和2.5克氯化钠。完全溶解后，增加150个毫升乙二醇的并调整到500毫升蒸馏水。储存在4℃的冷冻保护溶液。 </li><li>制备0.1M磷酸盐缓冲盐水（PBS）:加入18克NaCl的1升蒸馏水，同时搅拌。完全溶解后，加入磷酸钠缓冲液1升的0.2M。 </li><li>准备磷酸盐缓冲液添加0.3％的Triton X100（PBST）:加入3毫升三硝基甲苯X10的0 997毫升的PBS。 </li><li>制备的0.05M Tris-缓冲液（pH值为7.6）:添加3.03克的Tris-盐酸和0.70克亚磷酸三碱基至500ml，同时搅拌蒸馏水。 </li><li>准备2％山羊血清在PBST:一盘，准备加入30ml的解决方案。加入600微升山羊血清的至30ml PBST拌匀。加入每孔2.5毫升。 注意:使用血清必须来自用于二级抗体的产生种类</li><li>制备的兔多克隆抗体对c-fos蛋白（1:4000，SC-52）在PBST用牛血清白蛋白（0.25％）。对于一个盘子，准备加入30ml的解决方案。 75毫克牛血清白蛋白添加至30ml PBST拌匀。然后，添加7.5微升兔多克隆抗体对c-fos蛋白。 </li><li>制备生物素化的山羊抗兔抗体（1:500）在PBST与血清白蛋白的牛（0.25％）。对于一个盘子，准备加入30ml的解决方案。 75毫克牛血清白蛋白添加至30ml PBST拌匀。然后，添加60微升生物素化的山羊抗兔抗体。 </li><li>制备抗生物素蛋白 - 生物素 - 过氧化物酶复合物（1:250）在PBST（ABC溶液）。对于一个盘子，准备加入30ml的解决方案。加入试剂A的120微升和120微升试剂B〜30毫升PBST拌匀。 注:ABC的解决方案必须准备使用前至少30分钟。 </li><li>准备底物溶液。在即将使用之前，如下（为一个板）制备的溶液:添加20毫克硫酸镍铵和10mg 3.3二氨基四盐酸的至50ml Tris-缓冲液。过滤和避光。加入每孔2.0毫​​升。在剩余溶液（25毫升）加H 17微升2 O 2（H 2 O 2溶液H中2 O 30％）。加入每孔2.0毫​​升。 注意:适当的照顾应该用这些试剂时服用。手套和白大褂处理。 </li></ol> 2. c-fos INDUction和组织处理体内 注意:实验在大鼠（只SD）或小鼠（C57BL / 6）进行的。 <ol><li>放置的动物中有缺氧（O 2 8％的平衡N 2）或高二氧化碳（CO 2 4％，O 2 21％的平衡N 2）的气体混合物2小时（ 图2A）通风一个气密箱。 </li><li>麻醉用戊巴比妥腹膜内注射（100毫克/千克）动物。灌注动物transcardially用4％PFA 20。下一个化学罩护理和使用手套处理。 </li><li>灌注后，解剖脑20和浸入它在7ml 4％PFA中15毫升管在4℃下48小时。然后，从PFA取出大脑，并在冷冻保护溶液完全浸没了。商店在-18℃（ 图3）。由相应的废物处理处置PA动物的身体的其余部分的thway。 注意:冷冻保护是PFA固定和冷冻之间的工序。它减少了与其冷冻将是无定形形式的溶液冰晶的细胞的形成。在固定的样品必须在冷冻保护溶液（在步骤1.3中制备）为至少24小时，在4℃下进行浸渍。下化学罩护理和手套处理。 </li></ol> 3，c-fos诱导和组织处理离体 注意:实验在只新生大鼠或小鼠中进行。 <ol><li>如Suzue 19描述隔离中枢神经系统。放置在一个录音室，不断地与人工脑脊髓液在7.5毫升/分钟的速度在26°C 21（学联superfuse它:在MM:130.0氯化钠，5.4氯化钾，0.8 氯化钙 ，1.0 氯化镁 ，26.0的NaHCO 3，30.0 D-葡萄糖;用95％ 的 O 2和5放气饱和的 O 2，并调节至pH 7.4的％的CO 2）（ 图2B）。 </li><li>与低氧或通过酸化脑脊液（标准脑脊液用NaHCO 3降低到10.0毫脱氧脑脊液（用95％的N 2和5％的CO 2，pH 7.4的鼓泡相同的组成），用95％ 的 O 2和鼓泡取代脑脊液5％的CO 2）为高碳酸血症。申请的刺激30分钟（ 图2B）。 </li><li> 离体 ，诱导期后，在供以后使用（ 图3）冷冻保护溶液中2.5毫升4％PFA传送CNS新生小鼠或大鼠的一个2.5毫升管48小时，并储存于-18℃。 注意: 体外 ，这是没有必要灌注动物。先前的研究表明，小试样测量小于10毫米的直径可以通过简单的扩散22是固定的。渗透的引用率（距离，单位为mm，对于固定剂扩散到组织中），用于醛是2或3毫米/小时。 </l我&gt; </ol> 4.脑干切片注意:从该步骤到结束，该协议是严格用于体内和离体导入装置是相同的。 <ol><li>前切片，放置一个个体以及在一个12孔板插入并加入2.5倍毫升的PBS在每个孔中。 </li><li>使脑干的串行冠状切片（40微米）配有一个低温恒温器和收集它们在12孔板中。对于整个成年脑干，使约70切片（ 图3）。 注意:可以将部分的组织，从几个动物并联于一个板。 </li></ol> 5.免疫组织学程序（表1） 注意:在整个过程中，部分保留在井插入。 <ol><li> 1天-内源性过氧化物酶活破坏，非特异性结合位点的封锁，并与第一抗体孵育（图4） <ol><li>洗在室温10分钟，用0.​​1M PBS中的部分（2.5毫升，每孔）。重复3次。 </li><li>通过在RT与过氧化氢​​ħ孵育30分钟冠状切片抑制内源性过氧化物酶活2 O 2，3％在0.1M的PBS（2.5毫升，每孔）。 </li><li>洗部分在室温10分钟，用0.​​1M的PBS（2.5毫升，每孔）。重复3次。 </li><li>通过在RT用在PBST中的山羊血清（2％）温育1小时的冠状切片阻断非特异性结合位点。 </li><li> （:4000，SC-52，1）在PBST用牛血清白蛋白（0.25％）（2.5毫升，每孔）与抗C-FOS蛋白质的兔多克隆抗体孵育48小时的冠状切片在4℃下。 </li></ol></li><li> 第3天-孵育二次抗体和显色反应的发展（图4） <ol><li>洗部分10分钟，用PBS 0.1M的在RT（2.5毫升，每孔）。重复3次。 </li><li>孵育在PBST用牛血清白蛋白（0.25％）（2.5毫升，每孔）:在RT 1小时用生物素化的山羊抗兔抗体（500 1）冠状切片。 注意:研究者还可以使用耦合至荧光探针的二级抗体在这个阶段。在这种情况下，停止该协议在这个阶段，并用荧光显微镜直接检查。 </li><li>洗部分在室温10分钟用PBST（2.5毫升，每孔）。重复3次。 </li><li>孵育1小时的冠状切片与抗生物素蛋白 - 生物素 - 过氧化物酶复合物（1:250）在PBST（2.5毫升，每孔）。 </li><li>洗部分在室温10分钟用PBST（2.5毫升，每孔）。重复2次。 </li><li>洗部分在室温10分钟用0.05M Tris-缓冲液（2.5毫升，每孔）。重复2次。 </li><li>孵育0.02％3.3-二氨基联苯胺四盐酸盐，硫酸镍铵0.04％和0.01％的过氧化氢的冠状切片在0.05M Tris-缓冲液（pH7.6）中一个ŧRT（每孔2.0ml）中。在剩余溶液（25毫升）中添加17微升的 H 2 O 2（H 2 O 2溶液H中2 O 30％）（每孔2.0ml）中。 注:研究者应确定在光学显微镜下开发时间。然而，5至10分钟提供了良好的染色强度。 </li><li>当染色强度是最佳的（在光学显微镜下的控制，参见图5和图6），通过洗涤段在室温10分钟，用0.1M的PBS（2.5毫升，每孔）使反应停止。重复4次。然后，冲洗部分用蒸馏水。 </li></ol></li><li> 采样安装（处理与下化学罩护理和手套） <ol><li>山路段上连续幻灯片和风干。对于这一点，仔细涂抹几节rostro - 尾以便与幻灯片上的刷子。根据样品清楚标示的幻灯片。 </li><li>以绝对的人脱水cohol:浸入幻灯片在无水乙醇浴中在室温持续30秒。重复两次。 </li><li>清除用二甲苯:浸泡在二甲苯浴中的载玻片在室温3分钟。重复两次。 </li><li>盖玻片封固剂的幻灯片。对于这一点，应用5滴上滑动安装介质中，然后通过驱动出气泡申请玻璃盖。气干48小时。 </li></ol></li></ol> 6.数据和统计分析<ol><li>检查光镜下部分。目测计数，使用标准的地标23,24高倍率（200X）FLI神经元。在C-Fos的点状染色神经元细胞核本地化。 </li><li>对于每一个分析区域，计数每部分的c-FOS阳性细胞的数目和比较控制和刺激条件之间的平均数。根据数据的正常性，可以使用非配对学生t检验或Mann-Whitney检验。差异被认为若P显著0; 0.05。积FLI神经元上的图（放大100倍），使用的图管附连到显微镜用数码相机拍摄的（ 图5）的分布。 </li></ol>

<ol>
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The authors have nothing to disclose.

c-fos是一种立即早期基因，其产物的检测，所述c-fos蛋白，是古典用于识别体内 11,13,25,28参与具体呼吸道反应神经元群体和体外 16-18 27,32,33。 关键步骤的协议中 是在灌注一步小心。在4％PFA溶液必须很好制备和固定和后固定步骤必须足够长，以获得最佳的切片和染色。此外，启示是该过程的最重要的步骤;该染色的强度应该被控制，以避免背景噪声。 该技术与意义的优势相对于现有方法 尽管一些神经元似乎不表达c-fos基因5，本我的ThOD已被证明是有用的，以确定神经通路中化学挑战激活，例如，在呼吸节律适应。相比其他即早基因c-fos在缺乏刺激的低表达，允许试验情况6下神经元的活动更容易量化。 的C-FOS检测是蜂窝技术，表示为了分析涉及响应于若干刺激神经元群体中活性全球变化。使用电生理学方法，其中大部分的时间只涉及在一个确定的脑​​区域的细胞的数量少，c-fos的检测允许一个欣赏在大量细胞的活性的变化，因此，以限定有源神经元活性分析相比在整个中枢神经系统区域。 C-FOS检测与奔放和清醒动物容易兼容，这是不是ELECTR神经元的活动录制过程中的情况下ophysiology。这是一个优点，因为它避免了应力并用所获得的结果麻醉药干扰。 离体 ，我们使用了30分钟期间的刺激。这是以前表明这种刺激是足以在神经元水平34-36诱导c-fos表达的变化。刺激的五分钟足以诱导c-fos表达的变化，这是在15至20分钟，37的潜伏期后观察。在c-fos表达相关的增加被刺激的前10分钟内与在活动发生的变化。 该技术的局限性 刺激后，延迟是C-FOS的在细胞核中积累前必需的。约30分钟这个延迟对应于mRNA和蛋白质的合成。此产品的相对的结果的直接可能OBTA通过记录与电生理神经元的活动独立非执行董事。这可能会导致的关于特定和短暂刺激之后在神经元活动的快速变化的信息的损失。 为c-FOS蛋白质可具有90°的半衰期为100分钟4，其表达可以通过与动物的操纵，其约束，或者在其环境的变化相关联的外科手术或应力的影响。因此，有必要尽量减少能够诱导神经元活性的改变不相关的研究的刺激和生理刺激结束后立即执行组织取样操作。 修改和故障排除技术 在这个协议中，启示工序中使用含有3.3二氨基四盐酸，硫酸镍铵和过氧化氢的溶液中，但对于过氧化物的商业试剂盒可以人因此被用于此步骤。

所述C- fos基因在1980年1,2开始被确定为在第一时间和它的产物的特征在于在1984年为具有基因活化剂性能3,4-核蛋白质。它参与与神经刺激有关的长期机制。事实上，在神经元活动的变化导致第二信使信号级联诱导立即早期基因c-fos的表达，诱导产生的转录因子c-fos的。后者启动晚期基因的表达，从而参与神经系统的许多不同类型的刺激4适应性反应。因此，由于1980 5,6-结束时，C-FOS蛋白质检测已被经常用于研究的一般4和对中枢神经系统（CNS）的活性的外源因子对基因转录的影响为映射出神经通路参与不同的生理人的条件。 基础c-fos表达进行了研究不同物种包括老鼠，老鼠，猫，猴和人4。从而，其表达的动力学是相对众所周知的。转录激活是快速（5至20分钟）7,8，和mRNA的累积刺激9在发病后30和45分钟之间达到最大值并用12分钟的短半衰期下降。在c-fos蛋白合成如下mRNA的积累，可以通过免疫组化在20至90分钟后刺激6进行检测。 c-fos表达的分析在体内研究是经典用于鉴定参与低氧或高二氧化碳10-14的通气反应中枢性呼吸网络。最近，这个工具也用在体外脑干筹备探索呼吸中枢网络适应缺氧或Hypercapnia 15-18。事实上，这些制剂产生经典同化到呼吸中枢驱动器19有节奏的活动。因此，这种类型的制剂的具有被完全deafferented的优点，因此，对于c-fos表达的结果只反映中央的刺激所造成的后果，而不外 ​​围结构的任何干预。 在C-FOS检测可以通过免疫组化或immunohistofluorescence方法进行。间接免疫检测需要使用抗c-FOS一个初级抗体和针对在其中初级抗体产生的物质的第二抗体。对于免疫组织化学方法，所述二级抗体缀合与作用的基板（H 2 O 2为过氧化物酶）上的酶（过氧化物酶，例如）。酶反应的产物是由一个发色团（3.3二氨基联苯胺tetrahydrochlorid开发E），该污渍，并可以在光学显微镜下进行观察。该反应可以使用硫酸镍铵来增强。这些方法允许活性的神经元在不同生理挑战的检测，因此，鉴定和/或涉及的连续的生理反应外周和中枢途径的映射。

<table><tbody><tr><td>Cell culture plate 12-well</td><td>Costa</td><td>35/3</td><td></td></tr><tr><td>15 mm Netwell inserts with mesh polyester membrane</td><td>Corning</td><td>3477</td><td>The 15mm diameter well inserts have 74&amp;micro;m polyester mesh bottoms attached to polystyrene inserts</td></tr><tr><td>Primary antibody (rabbit polyclonal antibody against the c-Fos protein)</td><td>Santa Cruz Biotechnology</td><td>sc-52</td><td></td></tr><tr><td>Vectastain Elite ABC KIT&amp;nbsp;</td><td>Vector laboratories</td><td>PK-6101</td><td></td></tr><tr><td>(Rabbit IgG-secondary antibody)</td><td></td><td></td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma</td><td>71505</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S7907</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>NaOH 0.1N</td><td>Sigma</td><td>43617</td><td></td></tr><tr><td>Polyvinyl-Pyrrolidone</td><td>Sigma</td><td>PVP-360</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S7653</td><td></td></tr><tr><td>Ethylene-glycol</td><td>Sigma</td><td>33068</td><td></td></tr><tr><td>Triton X100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Trisma HCl</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>Trisma Base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>3.3-diaminobenzidine tetrahydrochloride&amp;nbsp;</td><td>Rockland</td><td>DAB50</td><td></td></tr><tr><td>Nickel ammonium sulphate</td><td>Alfa Aesar</td><td>12519</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>H1009</td><td></td></tr><tr><td>Xylene</td><td>Sigma</td><td>33817</td><td></td></tr><tr><td>Entellan Neo</td><td>Merck Millipore</td><td>107961</td><td></td></tr><tr><td>Slide&amp;nbsp;</td><td>Thermo-scientific</td><td>1014356190</td><td>Superfrost ultraplus</td></tr><tr><td>Cover glass</td><td>Thermo-scientific</td><td>Q10143263NR1</td><td>24 x 60mm</td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr></tbody></table>

the c-fos protein immunohistological detection: a useful tool as a marker of central pathways involved in specific physiological responses in vivo and ex vivo

许多研究努力查明并参与特定的生理法规的大脑区域。的原癌基因c-fos的 ，立即早期基因，是响应于各种刺激表达在神经元。蛋白质产物可与免疫组织化学技术导致使用的c-FOS检测到映射显示其活性的改变的神经元群体可容易地检测到。在这篇文章中，我们将重点放在参与通气适应低氧高碳酸血症或脑干神经元群体的鉴定。两种方法进行了描述，以确定参与神经元群体在体内在动物和体外在deafferented脑干的制剂。 在体内 ，动物暴露于高碳酸血症或缺氧的气体混合物。 体外 ，deafferented制剂灌流缺氧或高二氧化碳人工脑脊液。在这两种情况下，无论是控制在体内动物或电子邮件点¯x体内制剂常氧和碳酸血症的条件下保持。这两种方法的比较允许神经元激活即原点，测定外周和/或中枢， 在体内和体外 ，脑干，收集，固定，并切成段。一旦切片制备的C-FOS蛋白质的免疫组化检测，以便确定由低氧或高二氧化碳刺激活化细胞的脑干组作出。标记的细胞在脑干呼吸结构进行计数。相比于控制条件下，低氧或高二氧化碳增加在那些从而构参与中枢性呼吸驱动的适应的神经通路的几个具体脑干部位的c-FOS标记的细胞的数目。

的C-FOS检测是一个有用的工具，它允许如在体内 （ 图2A）低氧高二氧化碳或模仿这些条件下离体 （ 图2B）的情况下在特定条件下活化细胞的识别基团。 在体内 ，新生儿，青少年或成人啮齿动物被放置在气密箱中的气体环境是由一气体混合物为30至180分钟，13,25,26（ 图2A）精确限定的组合物连续地更新。随着刺激作用于全身，在C-FOS相关的变化可能反映了中央和周边激活途径。 体外 ，含延髓和脊髓deafferented制剂放置在不同级别的脑脊液持续灌流室O 2或pH值，以模拟低氧高碳酸血症或条件（ <stro纳克&gt;图2B）30分钟16-18,27。作为刺激仅作用在这些deafferented制剂，有可能得出结论，只有中央机制参与了细胞在c-FOS鉴定结构的活化。 中枢神经系统在体内通过体循环固定PFA，或者通过灌注，或通过浸渍体外 （ 图3）。由于这种差异，在固定剂扩散比较长的体内 体外 。因此，c-fos信号可能会受到影响，特别是在深层结构。然而，因为C-FOS分析是在控制之间的比较的方法始终执行和体内动物或离体的制剂的刺激，这种差异不会对结果从控制的一个严格比较干扰和刺激数据。在这之后，在CNS中冷冻保护，切片，以及从事即时munohistochemical过程（ 图3， 图4）。以这种方式，一些呼吸脑干区已在体内鉴定为下低氧或高二氧化碳挑战修改他们的活动，包括retrotrapezoid核（RTN），其为CO的主中心站点2化学感受10,13,28,29，该延髓腹外侧（VLM），其中包含吸气节律发生器13，连合和孤束核的中位数的（c / mNTS），这是在颈动脉体输入13,25,26,30（ 图5的投射区），和延髓缝核12,13。 离体 ，在deafferented的条件下，已经描述了以RTN和VLM的神经元来改变降低的 O 2 16-18中的条件下其的c-FOS免疫反应性。在C-FOS德tection也可以与其他免疫检测组合，以表征活化细胞14,31的表型<img alt="图1" src="/files/ftp_upload/53613/53613fig1.jpg" /> 图1.研究设计为使用C-FOS检测到地图中的呼吸调节适应低氧高碳酸血症或所涉及的神经结构，在中枢神经系统c-fos检测技术的年代步骤。中枢神经系统:中枢神经系统; IHC:免疫组化<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53613/53613fig2.jpg" /> 图2. C-FOS感应。（A） 在体内诱导协议。动物被放置在一个密闭Çhamber并暴露于空气（21％O 2; 79％N 2）高二氧化碳（21％ 的 O 2; 4％CO 2，75％N 2）或缺氧（10％ 的 O 2; 90％的N 2）气混合物2小时。 （B） &#160; 体外诱导方案。 CNS从0岁到4天的新生啮齿动物解剖出来。延髓和脊髓是在放大镜下分离出获得的筹备工作。它们在26±1℃下放置在一个腔室与腹面朝上，用人工脑脊液（pH7.4）中灌流。 体内建模缺氧刺激通过用鼓泡含有95％ 的 N 2和5％的CO 2缺氧的气体混合物还原的 O 2中的脑脊液的级分进行。模拟体内高碳酸血症刺激，使用酸学联从正常学联在不同的NaHCO 3浓度（减少）的条款执行。酸学联与O 2和adju饱和通过用95％ 的 O 2和5％CO 2鼓泡STED至pH 7.23。施加30分钟的每个刺激条件。学联:人工脑脊液<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53613/53613fig3.jpg" /> 图3.中枢神经系统采样。 在体内 ，中枢神经系统通过解剖后用4％多聚甲醛通过血管系统灌注固定剂的方法得到。 体外 ，中枢神经系统直接浸泡在4％低聚甲醛解剖后。接着，在CNS被浸没于冷冻保护溶液中冷冻保护和切成使用低温恒温器40微米厚的冠状切片。髓质rostro - 尾切片放入含有PBS befor一个12孔板Ë免疫程序。中枢神经系统:中枢神经系统; PBS:磷酸盐缓冲盐水<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53613/53613fig4.jpg" /> 图4.间接免疫程序。切片首先对c-fos蛋白兔多克隆一抗。之后，它们与针对第一抗体生物素化的山羊抗兔次级抗体处理。以下这个温育后，切片与抗生物素蛋白 - 生物素 - 过氧化物酶复合物，其紧密结合到第二抗体放大信号进行处理。事实上，复杂的包含几个过氧化物分子导致了高染色强度。最后，将切片用3.3 diamin的溶液中染色obenzidine四胺和硫酸镍铵，其产生的暗灰色的沉淀物在过氧化物酶的存在。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53613/53613fig5.jpg" /> 图5. 提交给 体内 缺氧对照小鼠和 延髓部分C-FOS阳性神经元 。（A） 延髓改编自Paxinos和Franklin 24（-7前囟门48毫米的一段绘图）。虚线区域delimitates 孤束核C / mNTS的合和中位数的部分。的框示出了由显微照片（B1和B2）中所示的结构。 （B）Photomicrographs在C / mNTS水平常氧（B1，控制，黑帧）或缺氧（B2。蓝帧）进行（X100）。黑箭头显示的c-FOS阳性细胞。 （C）直方图显示在C / mNTS水平常氧（黑条），或在缺氧条件下（蓝条）每节c-fos阳性细胞的平均数。值表示为平均值±标准差。 *表示常氧和缺氧值之间的差异显著-学生的配对t检验; *** P &lt;0.001。 CC:脊髓中央管; C / mNTS: 孤束核的合和中间部分<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/53613/53613table1.jpg" /> 表1.免疫组程序，在CF的年代步骤OS程序。 AB-Ⅰ，初级抗体; AB-II，二次抗体; BSA，牛血清白蛋白; DAB，3.3二氨基联苯胺四盐酸盐;镍，硫酸镍铵; PBS 0.1M，0.1M磷酸盐缓冲盐水（pH 7.4）中; PBST，0.1M磷酸盐缓冲盐水补充有0.3％的Triton X-100;血清，从用于二级抗体产生的物种的血清。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53613/53613table1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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Nanyang Technological University

Research

JoVE Journal

Biology

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

33.3K 次观看.  University Paris 13. Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

视频: The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

转录动力学的脑组织样本在行为经验综合分析

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

科研

行为学

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

癌症研究

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

神经科学

FIBS-enabled Noninvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be validated for predictive purposes. Ideally such systems would be low volume, which precludes sampling and destructive analyses when time course data are to be obtained. What is needed is an in situ monitoring tool which can report the necessary information in real-time and noninvasively. An interesting option is the use of fluorescent, protein-based in vivo biological sensors as reporters of intracellular concentrations. One particular class of in vivo biosensors that has found applications in metabolite quantification is based on F&#246;rster Resonance Energy Transfer (FRET) between two fluorescent proteins connected by a ligand binding domain. FRET integrated biological sensors (FIBS) are constitutively produced within the cell line, they have fast response times and their spectral characteristics change based on the concentration of metabolite within the cell. In this paper, the method for constructing Chinese hamster ovary (CHO) cell lines that constitutively express a FIBS for glucose and glutamine and calibrating the FIBS in vivo in batch cell culture in order to enable future quantification of intracellular metabolite concentration is described. Data from fed-batch CHO cell cultures demonstrates that the FIBS was able in each case to detect the resulting change in the intracellular concentration. Using the fluorescent signal from the FIBS and the previously constructed calibration curve, the intracellular concentration was accurately determined as confirmed by an independent enzymatic assay.

A description of how to calibrate F&#246;rster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented.&#160;The FIBS can be used to measure intracellular levels of metabolites noninvasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.

FIBS功能的无创性代谢轮廓

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

生物工程

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

使用免疫过氧化物酶和免疫荧光方法鉴定成年斑马鱼中的奥列辛和内他大麻素受体

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

生物化学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars and fats are mediated by their innate attractions as well as learned preferences. Brain dopamine, especially meso-limbic and meso-cortical projections from the ventral tegmental area (VTA), has been implicated in both of these unlearned and learned responses. The concept of distributed brain networks, wherein several sites and transmitter/peptide systems interact, has been proposed to mediate palatable food intake, but there is limited evidence empirically demonstrating such actions. Thus, sugar intake elicits DA release and increases c-fos-like immunoreactivity (FLI) from individual VTA DA projection zones including the nucleus accumbens (NAC), amygdala (AMY) and medial prefrontal cortex (mPFC) as well as the dorsal striatum. Further, central administration of selective DA receptor antagonists into these sites differentially reduce acquisition and expression of conditioned flavor preferences elicited by sugars or fats. One approach by which to determine whether these sites interacted as a distributed brain network in response to sugar or fat intake would be to simultaneous evaluate whether the VTA and its major mesotelencephalic DA projection zones (prelimbic and infralimbic mPFC, core and shell of the NAc, basolateral and central-cortico-medial AMY) as well as the dorsal striatum would display coordinated and simultaneous FLI activation after oral, unconditioned intake of corn oil (3.5%), glucose (8%), fructose (8%) and saccharin (0.2%) solutions. This approach is a successful first step in identifying the feasibility of using cellular c-fos activation simultaneously across relevant brain sites to study reward-related learning in ingestion of palatable food in rodents.

The goal of this study is to identify reward-related distributed brain networks by delineating a reliable immunohistological technique using cellular c-fos activation to measure simultaneous changes in dopamine pathways and terminal sites after novel ingestion of fat and sugar in rats.

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

在c-fos蛋白免疫组织学检测:一个有用的工具，中央通路在特定的生理反应所涉及的标记

摘要

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此视频中的章节

转录动力学的脑组织样本在行为经验综合分析

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

FIBS功能的无创性代谢轮廓

使用免疫过氧化物酶和免疫荧光方法鉴定成年斑马鱼中的奥列辛和内他大麻素受体

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

在c-fos蛋白免疫组织学检测:一个有用​​的工具，中央通路在特定的生理反应所涉及的标记

摘要

探索更多视频

此视频中的章节

转录动力学的脑组织样本在行为经验综合分析

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

慢性成人出生的嗅觉神经元的选择性病毒转导在体内 Optogenetic刺激

FIBS功能的无创性代谢轮廓

使用免疫过氧化物酶和免疫荧光方法鉴定成年斑马鱼中的奥列辛和内他大麻素受体

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

近红外荧光和高分辨率扫描测量啮齿类动物大脑中蛋白质的表达

在c-fos蛋白免疫组织学检测:一个有用的工具，中央通路在特定的生理反应所涉及的标记