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>在体内</em>和<em>离体</em
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The University Paris 13 supported this work. ASPT was supported by a University Paris 13 fellowship and the “Association Française 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).
Materials
| Cell culture plate 12-Well | Costa | 35/3 | |
| 15 mm Netwell inserts with mesh polyester membrane | Corning | 3477 | The 15mm diameter well inserts have 74µm polyester mesh bottoms attached to polystyrene inserts |
| primary antibody (rabbit polyclonal antibody against the c-Fos protein) | Santa Cruz Biotechnology | sc-52 | |
| Vectastain Elite ABC KIT | Vector laboratories | PK-6101 | |
| (Rabbit IgG-secondary antibody) | |||
| NaH2PO4*2H2O | Sigma | 71505 | |
| Na2HPO4 | Sigma | S7907 | |
| Paraformaldehyde | Sigma | P6148 | |
| NaOH 0.1N | Sigma | 43617 | |
| Polyvinyl-Pyrrolidone | Sigma | PVP-360 | |
| Sucrose | Sigma | S7903 | |
| NaCl | Sigma | S7653 | |
| Ethylene-glycol | Sigma | 33068 | |
| Triton X100 | Sigma | T8787 | |
| Trisma HCl | Sigma | T5941 | |
| Trisma Base | Sigma | T1503 | |
| 3.3-diaminobenzidine tetrahydrochloride | Rockland | DAB50 | |
| Nickel ammonium sulphate | Alfa Aesar | 12519 | |
| H2O2 | Sigma | H1009 | |
| Xylene | Sigma | 33817 | |
| Entellan Neo | Merck Millipore | 107961 | |
| Slide | Thermo-scientific | 1014356190 | Superfrost ultraplus |
| Cover glass | Thermo-scientific | Q10143263NR1 | 24 x 60mm |
| BSA | Sigma | A2153 |
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