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
Summary
在这里，我们提出了一个荧光活化细胞分选（FACS）协议来研究分子改建的Fos表达来自新鲜和冷冻的脑组织神经元歌舞团。使用冷冻组织可在多个会话许多脑区的FACS隔离，最大限度地利用宝贵的动物主题。
Abstract
神经可塑性和习得行为的分子变化的研究是从全脑区的研究转向的稀疏分布激活的神经元称为介导学协会神经歌舞团特定集合的研究。荧光活化细胞分选（FACS）最近已经成年大鼠脑组织进行了优化，并允许使用抗神经元的NeuN标记和Fos蛋白，强烈地激活的神经元的标志物激活的神经元的隔离。到现在为止，Fos蛋白表达的神经元和其他细胞类型，从新鲜组织，从而享有很长的处理天脑样品的允许号码非常有限的冗长和复杂的行为过程之后进行评估隔离。在这里，我们发现，Fos蛋白表达来自背侧纹状体神经元和Fos蛋白的mRNA产率分别为新鲜解剖组织和组织在-80ºC冷冻3之间相似- 21天。另外，我们证实了表型所述的NeuN阳性和的NeuN阴性分选的细胞通过神经元的评估基因表达的（ 的NeuN），星形细胞（GFAP），少突胶质细胞（Oligo2）和microgial（Iba1）标记物，这表明冷冻组织也可用于的FACS分离的神经胶质细胞类型。总体而言，它是可以收集，解剖和冷冻多个FACS会话脑组织。这最大化从有价值动物受试者已经经常发生长和复杂的行为过程获得的数据量。
Introduction
学习过程中，动物形成复台高度特异性的刺激之间的关联。此高分辨率信息被认为是由稀疏分布的神经元称为神经合奏特定图案内的改变进行编码。神经乐团最近已确定了的立即早期基因（即早基因）如Fos的 ， 圆弧和Zif268及其蛋白质产物在神经元的行为或线索暴露在强烈激活的诱导。 fos基因表达的神经元尤其已经示出在背景和线索特定习得行为1-4发挥因果作用。因此，这些被激活的Fos表达神经元内独特的分子neuroadaptations是编码了解到，在正常的学习和正常的学习障碍形成的协会，如成瘾和创伤后应激障碍（PTSD）5的神经机制的热门人选。
荧光李宗红激活细胞分选（FACS）最近允许的Fos蛋白表达的神经元内独特的分子neuroadaptations分析。流式细胞术和细胞分选在1960 6,7被开发根据它们的光散射和免疫特征来表征和分离细胞，并且早已在免疫学和癌症研究中使用。然而流式细胞仪和FACS需要解离的单细胞难以从成人脑组织得到。 FACS最早是用来分离和分析绿色荧光蛋白（GFP）-expressing从没有要求抗体标记8,9转基因小鼠纹状体神经元。我们开发了一种基于抗体的FACS方法10来隔离并在的Fos表达由药物和/或暗示在野生型动物中11-15激活的神经元评估分子改变。在该方法中，神经元被标以对一般的神经元标记物的NeuN的抗体，而强烈激活的神经元标记与抗Fos蛋白的抗体。虽然我们最初的方法所要求达到10只，每个样品新鲜组织池，该协议的后续修改允许从单个老鼠13-15的Fos表达神经元和定量聚合酶链反应（定量PCR）分立的大脑区域的分析，流式细胞仪分离。总体而言，在Fos蛋白表达的成瘾研究12,14,15各种上下文和提示激活行为的过程中激活的神经元中发现独特的分子变化。
与新鲜组织进行的FACS的一个主要的后勤问题是，它需要一整天以解离通过FACS组织和处理。此外，只有大约四个样品每天可处理。这通常意味着只有一个大脑区域可从各脑进行评估和剩余的脑区域都被丢弃。这对于低吞吐量行为程序，如自我施用和消光TRAI一个主要问题宁认为需要手术治疗和强化训练几个星期。此外，在测试当天长期和复杂的行为过程使得难以在同一天进行的FACS。这将是一个显著优点是能够从动物行为测试后，立即冻结的大脑，然后在研究者的选择的不同的时间从一个或多个脑区域隔离的Fos表达神经元。
在这里，我们证明，我们的FACS协议可以用于分离从新鲜和冷冻的脑组织的Fos表达神经元（和其它细胞类型）。作为一个例子，我们分离的Fos表达后急性注射甲基苯丙胺，并从幼稚大鼠不打针（对照条件）从纹状体神经元。然而，该FACS协议可以按照任何行为或药物治疗中使用。我们的样品的后续的qPCR分析表明，从这些类型的细胞的基因表达可以与西米进行评估从新鲜和冷冻组织征地拆迁的效率。
Protocol
Representative Results
Discussion
FACS可用于从新鲜或冷冻的成人脑组织的神经元和其他细胞类型进行排序。如在引言中提到的，使用冷冻组织的能力允许从已经历复杂和长期行为的程序，如在成瘾研究自我施用和复发的研究的动物样品的最佳利用。这些行为过程通常需要1 – 2小时或更长的时间，并且要求所有动物（10 – 20个）上13,18在同一天进行测试。它需要〜4小时处理4新鲜解剖脑组织样本，其包括脑解剖，细胞解离，?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
This work was supported by the NIDA Intramural Research Program (Bruce T. Hope, Yavin Shaham). F.J.R. was supported by an appointment to the NIDA Research Participation Program sponsored by the National Institutes of Health and administered by the Oak Ridge Institute for Science and Education, and received additional financial support from a Becas-Chile scholarship managed by CONICYT and the Universidad de los Andes, Santiago, Chile. The Johns Hopkins FACS Core facility was supported by Award P30AR053503 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health.
Materials
| Brain matrix | CellPoint Scientific | 69-2160-1 | to obtain coronal brain slices |
| Hibernate A low fluorescence | Brain Bits | HA-lf | Buffer A' in the protocol is used for processing tissue and cells from the dissociation to fixation steps |
| Accutase | Millipore | SCR005 | Enzyme solution' in the protocol is used for enzymatic digestion of tissue prior to trituration |
| Protein LoBind Tube 1.5 mL, PCR clean | Eppendorf | 22431081 | to prevent cell lost during the protocol |
| Cell Strainer, 40 µm | BD Falcon | 352340 | to filter cell suspension |
| Cell Strainer, 100 µm | BD Falcon | 352360 | to filter cell suspension |
| Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap | BD Bioscience | 352235 | to filter cell suspension before passing though the flow cytometer |
| Pasteur Pipet, Glass | NIH supply | 6640-00-782-6008 | to do tissue trituration |
| Milli-Mark Anti-NeuN-PE, Clone A60 (mouse monoclonal) antibody | EMD Millipore | FCMAB317PE | antibody to detect neurons |
| Phospho-c-Fos (Ser32) (D82C12) XP Rabbit (Alexa Fluor 647 Conjugate) | Cell Signaling Technology | 8677 | antibody to detect Fos-expressing cells |
| DAPI | Sigma | D8417 | to label nuclei |
| PicoPure RNA Isolation Kit | Applied Biosystems. | KIT0204 | The kit includes the RNA extraction buffer for step 6.14. It is used to collect sorted cells |
| Superscript III first strand cDNA synthesis system | Invitrogen | 18080-051 | to synthesize cDNA from RNA |
| TaqMan PreAmp Master Mix | Applied Biosystems. | 4391128 | to do targeted-preamplification from cDNA |
| TaqMan Advance Fast Master | Applied Biosystems. | 4444963 | to do PCR using TaqMan probes |
| Fos TaqMan probe | Applied Biosystems. | Rn00487426_g1 | TaqMan probe/primers |
| NeuN TaqMan probe | Applied Biosystems. | CACTCCAACAGCGTGAC | nucleotide sequence for neuronal gene marker |
| NeuN Forward primer | Applied Biosystems. | GGCCCCTGGCAGAAAGTAG | nucleotide sequence for neuronal gene marker |
| NeuN Reverse primer | Applied Biosystems. | TTCCCCCTGGTCCTTCTGA | nucleotide sequence for neuronal gene marker |
| Gfap TaqMan probe | Applied Biosystems. | Rn00566603_m1 | TaqMan probe/primers for astrocityc gene marker |
| iba-1 TaqMan probe | Applied Biosystems. | Rn00574125_g1 | TaqMan probe/primers for microglya gene marker |
| Gapdh TaqMan probe | Applied Biosystems. | CTCATGACCACAGTCCA | nucleotide sequence for reference/housekeeping gene |
| Gapdh Forward primer | Applied Biosystems. | GACAACTTTGGCATCGTGGAA | nucleotide sequence for reference/housekeeping gene |
| Gapdh Reverse primer | Applied Biosystems. | CACAGTCTTCTGAGTGGCAGTGA | nucleotide sequence for reference/housekeeping gene |
| Oligo2 TaqMan probe | Applied Biosystems. | Rn01767116_m1 | TaqMan probe/primers for oligodendrocytic gene marker |
| P1000 pipettor | Rainin | 17014382 | It is refered to as the pipette with a large tip diameter in steps 3.1 and 3.3 for mild tissue trituration and step 6.7 to resuspend cells |
| 7500 Fast Real-time PCR system | Applied Biosystems. | 446985 | for quantitative PCR |
| FACSAria I Cell Sorter | BD Biosciences | for FACS sorting |
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