结合双荧光<em>原位</em>杂交免疫标记为三个基因在小鼠大脑切片中表达的检测
Summary
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.
Abstract
在不同类型的脑细胞如神经元，星形胶质细胞，少突胶质细胞，少突胶质细胞前体和小胶质细胞的基因表达的检测，可通过缺乏免疫染色特定一次或二次抗体的受到阻碍。这里，我们描述一个协议，以检测在同一脑切片三种不同的基因使用原位杂交双荧光表达两个基因特异性探针，接着用高特异性针对由第三基因编码的蛋白质的抗体的免疫染色。所述Aspartoacyclase（ASPA）基因，其中突变可导致一种罕见的人类白质疾病-卡纳疾病-被认为在少突胶质细胞和小胶质细胞，但不是在星形胶质细胞和神经元中表达。然而，在大脑ASPA的精确表达模式还没有被确立。该协议使我们能够确定ASPA在成熟的少突胶质细胞的一个子集表示第二，可以普遍适用于广泛的基因表达模式的研究。
Introduction
神经胶质细胞，这是最丰富的细胞在中枢神经系统（CNS），包括少突胶质细胞（CNS的髓鞘形成细胞），少突胶质细胞前体（OP的，也被称为“NG2细胞”），星形胶质细胞和小胶质细胞。有在神经胶质细胞和他们的潜在作用的功能越来越大的兴趣在神经性疾病1。例如，卡纳万病（CD）是一种遗传性神经退行性疾病与海绵状脑白质营养不良和神经元的逐渐丧失起步阶段起步早，一般10年之前的2,3岁时导致死亡。在Aspartoacyclase（ASPA）基因突变导致急剧减少ASPA活动4 CD已经确定。 ASPA是催化N-乙酰（NAA）的脱乙酰酶，高度集中在脑的分子，产生乙酸和天冬氨酸5-7。许多CD患者表现出由于缺乏交流ASPA更高水平的NAA拉了一。一些研究推测NAA衍生醋酸可以是脂肪酸/在大脑中脂质的主要来源发育期间和CD可能导致引起NAA未能发展被分解-3,5,6-期间减少髓鞘合成。
ASPA在大脑的肾脏，肝脏和白质主要存在，并给予ASPA的在光盘中的重要作用，在大脑中的这种酶的细胞表达，研究了几个实验室。通过查看在大脑ASPA酶活性，以前的研究发现，脑发育过程中的增加ASPA活性平行髓鞘8-10的时间过程。在细胞水平，对酶的活性，以及原位杂交（ISH）和免疫组织化学（IHC）测定分析表明，ASPA主要表达在大脑，但不是在神经元或胶质细胞11-16少突胶质细胞。一些研究发现，ASPA也说不定在小神经胶质细胞在CNS 12,14来表示。在有机磷农药对ASPA表达到目前为止的数据是有限的。根据最近的研究，其中在小鼠大脑皮层包括神经元，星形胶质细胞，有机磷农药，新形成的少突胶质细胞，髓鞘少突胶质细胞，小胶质细胞，内皮细胞和周细胞不同的细胞类型的转录由RNA测序17进行了分析，ASPA专门在少突胶质细胞表达，特别是在髓鞘少突胶质细胞（http://web.stanford.edu/group/barres_lab/brain_rnaseq.html）。尽管在大脑ASPA表达模式这些研究中，一些不确定因素仍然存在。
不同的技术可用于研究基因表达模式。 IHC是用于组织切片检测基因表达的功能的产品（ 即，蛋白质）通常使用的方法。尽管它的巨大效用，这种技术具有局限性，因为它的应用和特异性对象TØ所需的抗体的可用性和特异性。通过比较，ISH具有能够在mRNA水平发现任何基因的表达的优点。然而，它可以是在技术上具有挑战性的，以便定位一个基因的表达，以特定的细胞类型，同时使用几种探针。在这篇文章中，我们描述了一个协议， 原位杂交蛋白质的免疫标记荧光双结合的荧光RNA。我们使用这组技术来检查电力局在小鼠大脑的表达模式。此方法允许使用共聚焦显微镜的基因表达的精确研究。
Protocol
Representative Results
Discussion
该协议提供了一个一步一步的过程原位杂交双RNA后免疫。我们已经用这种协议来确认电力局在几个脑区表达成熟的少突胶质细胞。
这个多步过程有可能影响灵敏度，应该避免了许多潜在的隐患。首先，用于转录反应的所有解决方案和存储缓冲器需要是无RNase。第二，cDNA的模板的选择是重要的，我们在长度大约1kb的偏爱模板。 体外转录之前，有必要进行清?…
Declarações
The authors have nothing to disclose.
Acknowledgements
Work in the authors’ laboratories was supported by the UK Biotechnology and Biological Sciences Research Council (BB/J006602/1 and BB/L003236/1), the Wellcome Trust (WT100269MA) and the European Research Council (ERC, “Ideas” Programme 293544). SJ was supported by an EMBO long-term fellowship. The authors thank Stephen Grant for his technical assistance.
Materials
| QIAprep® Miniprep | Qiagen | 27104 | |
| Deionized formamide | Sigma | F9037 | for ISH blocking buffer |
| Sodium chloride | Sigma | S3014 | |
| Trizma Base | Sigma | T1503 | |
| Hydrochloric acid | VWR International | 20252.290 | |
| Sodium phosphate monobasic anhydrous | Sigma | S8282 | |
| Sodium phosphate dibasic dihydrate | Sigma | 30435 | |
| Yeast tRNA | Roche | 10109495001 | |
| 50x Denhardt's solution | Life Technologies | 750018 | |
| Dextran sulfate | Sigma | D8906 | |
| Aspa cDNA clone | Source Bioscience | IRAVp968C0654D | |
| SalI | New England Biolabs | R0138 | |
| Sodium acetate | Sigma | S2889 | |
| Equilibrated phenol | Sigma | P4557 | |
| Chloroform | Sigma-Aldrich | C2432 | |
| Isoamyl alcohol | Aldrich | 496200 | |
| Ethanol | VWR International | 20821.321 | |
| T7 RNA polymerase | Promega | P4074 | |
| Transcription buffer | Promega | P118B | |
| 100mM DTT | Promega | P117B | |
| UTP-DIG NTP mix | Roche | 11277073910 | |
| Rnasin | Promega | N251B | |
| Paraformaldehyde | Sigma | P6148 | |
| Filter paper | Fisher scientific | 005479470 | |
| Sucrose | Sigma | 59378 | |
| Diethyl pyrocarbonate | Sigma | D5758 | |
| Pentobarbitone | Animalcare Ltd | BN43054 | |
| Dissecting scissors | World Precision Instruments | 15922 | |
| 25 gauge needle | Terumo | 300600 | |
| Peristaltic pump | Cole-Parmer Instrument Co. Ltd | WZ-07522-30 | |
| Iris scissors | Weiss | 103227 | |
| No.2 tweezers | World Precision Instruments | 500230 | |
| Coronal Brain Matrix | World Precision Instruments | RBMS-200C | |
| Razor blade | Personna Medical | PERS60-0138 | |
| OCT medium | Tissue tek | 4583 | |
| Cryostat/microtome | Bright | ||
| Superfrost plus slides | Thermo Scientific | J1800AMNZ | |
| Sodium citrate | Sigma | S4641 | for 65°C wash buffer |
| Formamide | Sigma-Aldrich | F7503 | |
| Tween-20 | Sigma-Aldrich | P1379 | |
| Coverslips | VWR International | 631-0146 | |
| Coplin Jar | Smith Scientific Ltd | 2959 | |
| Blocking reagent | Roche | 11096176001 | |
| Heat-inactivated sheep serum | Sigma | S2263 | |
| Hydrophobic pen | Cosmo Bio | DAI-PAP-S | 1:500 |
| α-FITC POD-conjugated antibody | Roche | 11426346910 | |
| TSA™ Plus Fluorescein System | Perkin Elmer | NEL741001KT | 1:1500 |
| α-DIG AP-conjugated | Roche | 11093274910 | |
| Fast red tablets | Roche | 11496549001 | |
| .22µM filter | Millex | SLGP033RS | |
| α-Olig2 Rabbit antbody | Millipore | AB9610 | |
| Alexa Fluor® 647-conjugated α-rabbit antibody | Life technologies | A-31573 | 1:1000 |
| bisBenzimide H 33258 | sigma | B2883 | |
| Mounting medium | Dako | S3023 | |
| Leica SP2 confocal microscope | Leica |
Referências
- Lobsiger, C. S., Cleveland, D. W. Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease. Nat Neurosci. 10 (11), 1355-1360 (2007).
- Baslow, M. H. N-acetylaspartate in the vertebrate brain: metabolism and function. Neurochem Res. 28 (6), 941-953 (2003).
- Hoshino, H., Kubota, M. Canavan disease: clinical features and recent advances in research. Pediatr Int. 56 (4), 477-483 (2014).
- Kaul, R., Gao, G. P., Balamurugan, K., Matalon, R. Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease. Nat Genet. 5 (2), 118-123 (1993).
- Divry, P., Mathieu, M. Aspartoacylase deficiency and N-acetylaspartic aciduria in patients with Canavan disease. Am J Med Genet. 32 (4), 550-551 (1989).
- Bartalini, G., et al. Biochemical diagnosis of Canavan disease. Childs Nerv Syst. 8 (8), 468-470 (1992).
- Moffett, J. R., Ross, B., Arun, P., Madhavarao, C. N., Namboodiri, A. M. N-Acetylaspartate in the CNS: from neurodiagnostics to neurobiology. Prog Neurobiol. 81 (2), 89-131 (2007).
- D’Adamo, A. F., Smith, J. C., Woiler, C. The occurrence of N-acetylaspartate amidohydrolase (aminoacylase II) in the developing rat. J Neurochem. 20 (4), 1275-1278 (1973).
- Bhakoo, K. K., Craig, T. J., Styles, P. Developmental and regional distribution of aspartoacylase in rat brain tissue. J Neurochem. 79 (1), 211-220 (2001).
- Sommer, A., Sass, J. O. Expression of aspartoacylase (ASPA) and Canavan. Gene. 505 (2), 206-210 (2012).
- Klugmann, M., et al. Identification and distribution of aspartoacylase in the postnatal rat brain. Neuroreport. 14 (14), 1837-1840 (2003).
- Madhavarao, C. N., et al. Immunohistochemical localization of aspartoacylase in the rat central nervous system. J Comp Neurol. 472 (3), 318-329 (2004).
- Hershfield, J. R., et al. Aspartoacylase is a regulated nuclear-cytoplasmic enzyme. Faseb J. 20 (12), 2139-2141 (2006).
- Moffett, J. R., et al. Extensive aspartoacylase expression in the rat central nervous system. Glia. 59 (10), 1414-1434 (2011).
- Kirmani, B. F., Jacobowitz, D. M., Kallarakal, A. T., Namboodiri, M. A. Aspartoacylase is restricted primarily to myelin synthesizing cells in the CNS: therapeutic implications for Canavan disease. Brain Res Mol Brain Res. 107 (2), 176-182 (2002).
- Kirmani, B. F., Jacobowitz, D. M., Namboodiri, M. A. Developmental increase of aspartoacylase in oligodendrocytes parallels CNS myelination. Brain Res Dev Brain Res. 140 (1), 105-115 (2003).
- Zhang, Y., et al. An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci. 34 (36), 11929-11947 (2014).
