斑马鱼胚胎和幼虫的整个山免疫性化学
Summary
在这里，我们提出了一个方案，荧光抗体介导检测蛋白质在斑马鱼胚胎和幼虫的整个制剂。
Abstract
免疫组织化学是一种广泛使用的技术，用于在正常发育和疾病状态下探索蛋白质表达和定位。尽管许多免疫组织化学方案已针对哺乳动物组织和组织部分进行了优化，但这些协议通常需要对非哺乳动物模型生物体进行修改和优化。斑马鱼越来越多地作为基础、生物医学和转化研究的模型系统，用于研究发育过程的分子、遗传和细胞生物学机制。斑马鱼作为模型系统具有许多优点，但也需要经过修改的技术来优化蛋白质检测。在这里，我们提供斑马鱼胚胎和幼虫全装荧光免疫基化学方案。该协议还描述了可以使用的几种不同的安装策略，并概述了每种策略提供的优缺点。我们还描述了对该协议的修改，以允许检测整个安装组织中的染色体基质和切片幼虫组织的荧光检测。该议定书广泛适用于许多发育阶段和胚胎结构的研究。
Introduction
斑马鱼（达尼奥再鱼）已成为研究生物过程的有力模型，原因包括发电时间短、发展迅速、遗传技术的可及性等。因此，斑马鱼通常用于高通量小分子屏幕的毒理学研究和药物发现。Zebrafish 也是研究发育过程的一个有吸引力的模型，因为单个雌性可以一次正常生产 50-300 个卵子，而光学清晰的胚胎在外部发育，从而能够有效地可视化发育过程。然而，由于在建立反向遗传技术方面的挑战，早期研究主要依靠使用N-乙基-N-硝基苏雷亚（ENU）或其他诱变基因的正向遗传屏幕。大约20年前，斑马鱼首次使用变形金刚来敲除靶向基因1。形态诺是一种小的反义寡核苷酸，在早期发育阶段将微注射到胚胎后抑制目标mRNA的转化。形态软糖的一个主要弱点是，当细胞分裂时，它们被稀释，通常在受精后72小时（hpf）时失去效力。虽然形态动物仍然是斑马鱼基因破坏的有力工具，但转录活化样核酸酶（TALEN）、锌指核酸酶（ZFNs）和定期聚因间隔短回溯重复（CRISPRs）最近被用于直接靶向斑马鱼基因组2，3。这些反向遗传策略，结合正向遗传学和高通量屏幕，建立了斑马鱼作为研究基因表达和功能的强大模型。
研究基因功能的能力通常需要评估基因或基因产物表达的时空分布。在发育早期可视化这种表达模式的两种最常用的技术是原位杂交（ISH）和全装载免疫组织化学（IHC）。原位杂交于1969年首次开发，依靠使用标记的反义RNA探针来检测生物体4中的mRNA表达。相反，标记抗体用于免疫组织化学，以可视化蛋白质表达。标记蛋白质进行检测的想法可追溯到20世纪30年代的5世纪，1941年，第一个IHC实验发表于1941年，当时FITC标记的抗体被用来检测受感染组织中的致病细菌。ISH和IHC在随后几十年中已经发展并显著改善，现在都常规地用于分子和诊断研究实验室7，8，9，10，11。虽然这两种技术都有优点和缺点，但 IHC 比 ISH 具有若干优点。实际上，IHC 比 ISH 耗费少得多，并且根据原抗体的成本通常较低。此外，mRNA表达并不总是蛋白质表达的可靠指标，正如在小鼠和人类中已经证明的，只有大约三分之一的蛋白质丰度变异可以通过mRNA丰度12来解释。因此，IHC 是确认 ISH 数据的重要补充，如果可能的话。最后，IHC可以提供亚细胞和共同本地化数据，这些数据不能由ISH13、14、15确定。在这里，我们描述了一个分步法，通过免疫性化学在整座斑马鱼胚胎和幼虫中可靠地检测蛋白质。这项技术的目的是确定整个胚胎中感兴趣的蛋白质的时空表达。该技术利用抗原特异性原抗体和荧光标记的二级抗体。该协议易于适应，适用于滑动安装的组织部分，并与致变色基质一起用于荧光。使用这个协议，我们证明，除了乙酰胆碱受体外，开发斑马鱼骨骼肌表达的有益性谷氨酸受体。NMDA型谷氨酸受体亚单位在23 hpf的纵向肌肉上可检测到。
Protocol
本议定书中描述的斑马鱼繁殖成人和胚胎的手术程序得到了默里州立大学机构动物护理和使用委员会的批准。 1. 胚胎收集和固定 通过将成年斑马鱼混合性对或组放入装有系统水的网状或槽内衬的水箱中，准备产卵池。 在亮起灯时，将产卵罐水更换为新鲜系统水，以清除粪便。使用 14 小时/10 小时暗循环，上午 9 点亮起灯。 一旦产卵，让大人回到家里的…
Representative Results
全装免疫组织化学利用抗体检测完整动物中蛋白质表达的空间模式。免疫组织化学的基本工作流程（如图1所示）包括繁殖斑马鱼、培育和制备胚胎、阻断非特异性抗原、使用抗原特异性原抗体靶向感兴趣的蛋白质、检测带有标记的次级抗体的初级抗体、安装标本和记录表达。 全山免疫性化学是研究斑马?…
Discussion
免疫组织化学是一种多功能的工具，可用于描述生物体中几乎所有感兴趣的蛋白质的时空表达。免疫组织化学用于各种组织和模型生物体。该协议已针对斑马鱼的使用进行了优化。不同物种的免疫组织化学可能需要不同的固定和处理技术，根据物种和内源性过氧化物酶的存在阻塞溶液，以及由于组织的厚度和组成而孵育的时间。斑马鱼中的IHC在增进我们对癌症24、代谢性疾病<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
国家卫生研究院的资助赠款8P20GM103436 14。
Materials
| Agarose | Fisher Scientific | BP160-100 | |
| Aluminum foil, heavy duty | Kirkland | Any brand may be substituted | |
| Anti-NMDA antibody | Millipore Sigma | MAB363 | |
| Anti-phospho-Histone H3 (Ser10), clone RR002 | Millipore Sigma | 05-598 | |
| Anti-pan-AMPA receptor (GluR1-4) | Millipore Sigma | MABN832 | |
| Bovine serum albumin (BSA) | Fisher Scientific | BP1600-100 | |
| Calcium Nitrate [Ca(NO3)2] | Sigma Aldrich | C4955 | |
| Centrifuge tubes, 1.5 mL | Axygen | MCT150C | |
| Clear nail polish | Sally Hanson | Any nail polish or hardener may be subsituted | |
| Depression (concavity) slide | Electron Miscroscopy Sciences | 71878-01 | |
| Diaminobenzidine | Thermo Scientific | 1855920 | |
| Embryo medium, Danieau, 30% | 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgS04, 0.18 mM Ca(NO3)2, 1.5 mM HEPES in ultrapure water. | ||
| Embryo medium, E2 | 7.5 mM NaCl, 0.25 mM KCl, 0.5 mM MgSO4, 75 uM KH2PO4, 25 uM Na2HPO4, 0.5 mM CaCl2, 0.35 mM NaHCO3, 0.5 mg/L methylene blue | ||
| Floating tube holder | Thermo Scientific | 59744015 | |
| Fluorescence compound microscope | Leica Biosystems | DMi8 | |
| Fluorescence stereomicroscope | Leica Biosystems | M165-FC | |
| Glass coverslips 18 x 18 | Corning | 284518 | |
| Glass coverslips 22 x 60 | Thermo Scientific | 22-050-222 | |
| Glass slides | Fisher Scientific | 12-544-4 | |
| Glycerol | Fisher Scientific | BP229-1 | |
| Goat anti-mouse IgG Alexa 488 | Invitrogen | A11001 | |
| HEPES solution | Sigma Aldrich | H0887 | |
| Humid chamber with lid | Simport | M920-2 | |
| Hydrogen peroxide, 30% | Fisher Scientific | H325-500 | |
| Immunedge pap pen | Vector labs | H-4000 | |
| Insect pins, size 00 | Stoelting | 5213323 | |
| Magnesium Sulfate (MgSO4 · 7H2O) | Sigma Aldrich | 63138 | |
| Mesh strainer | Oneida | Any brand may be substituted | |
| Methanol | Sigma Aldrich | 34860 | |
| Methylene blue | Sigma Aldrich | M9140 | |
| Micro-tube cap lock | Research Products International | 145062 | |
| Microwave oven | Toastmaster | ||
| Mouse IgG | Sigma Aldrich | I8765 | |
| Normal goat serum | Millipore Sigma | S02L1ML | |
| Nutating mixer | Fisher Scientific | 88-861-044 | |
| Paraformaldehyde | Fisher Scientific | 04042-500 | |
| Pasteur pipettes | Fisher Scientific | 13-678-20C | |
| PBTriton | 1% TritonX-100 in 1x PBS | ||
| Permount mounting medium | Fisher Chemical | SP15-500 | |
| Petri dish (glass) | Pyrex | 3160100 | |
| Petri dish (plastic) | Fisher Scientific | FB0875713 | |
| 1-phenyl 2-thiourea | Acros Organics | 207250250 | |
| Phosphate buffered saline (PBS), 10x, pH 7.4 | Gibco | 70011-044 | |
| Phosphate buffered saline (PBS), 1x | 1x made from 10x stock diluted in dH2O | ||
| Potassium Chloride (KCl) | Sigma Aldrich | P9333 | |
| Potassium Hydroxide (KOH) | Fisher | P250-500 | |
| Potassium Phosphate Monobasic (KH2PO4) | Sigma Aldrich | P5655 | |
| Pronase | Sigma Aldrich | 10165921001 | |
| Proteinase K | Invitrogen | AM2544 | |
| Sodium Chloride (NaCl) | Sigma Aldrich | S7653 | |
| Sodium Phosphate Dibasic (Na2HPO4) | Sigma Aldrich | S7907 | |
| Spawning tank with lid and insert | Aquaneering | ZHCT100 | |
| SuperBlock PBS | Thermo Scientific | 37515 | |
| Superfrost + slides | Fisher Scientific | 12-550-15 | |
| Superglue gel | 3M Scotch | ||
| TNT | 100 mM Tris, pH 8.0; 150 mM NaCl; 0.1% Tween20; made in dH2O | ||
| Transfer pipette | Fisher | 13-711-7M | |
| Trichloracetic Acid (Cl3CCOOH) | Sigma Aldrich | T6399 | |
| Tris Base | Fisher Scientific | S374-500 | |
| TritonX-100 | Sigma Aldrich | T9284 | |
| Tween20 | Fisher Scientific | BP337-500 | |
| Ultrafine forceps | Fisher Scientific | 16-100-121 | |
| Water, ultrapure/double distilled | Fisher Scientific | W2-20 |
References
- Nasevicius, A., Ekker, S. C. Effective targeted gene ‘knockdown’ in zebrafish. Nature Genetics. 26 (2), 216-220 (2000).
- Gaj, T., Gersbach, C. A., Barbas, C. F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology. 31 (7), 397-405 (2013).
- Irion, U., Krauss, J., Nusslein-Volhard, C. Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 system. Development. 141 (24), 4827-4830 (2014).
- van der Ploeg, M. Cytochemical nucleic acid research during the twentieth century. European Journal of Histochemistry. 44 (1), 7-42 (2000).
- Marrack, J. Nature of Antibodies. Nature. 133 (3356), 292-293 (1934).
- Coons, A. H., Creech, H. J., Jones, R. N. Immunological properties of an antibody containing a fluorescent group. Proceedings of the Society for Experimental Biology and Medicine. 47 (2), 200-202 (1941).
- Lopez, M. E. Combined in situ Hybridization/Immunohistochemistry (ISH/IH) on Free-floating Vibratome Tissue Sections. Bio-protocol. 4 (18), (2014).
- Morimoto, M., Morita, N., Ozawa, H., Yokoyama, K., Kawata, M. Distribution of glucocorticoid receptor immunoreactivity and mRNA in the rat brain: an immunohistochemical and in situ hybridization study. Neuroscience Research. 26 (3), 235-269 (1996).
- Newton, S. S., Dow, A., Terwilliger, R., Duman, R. A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen, cryocut mouse brain sections. Brain Research Protocols. 9 (3), 214-219 (2002).
- Corthell, J. T., Corthell, J. T. . Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. , 105-111 (2014).
- Corthell, J. T., Corthell, J. T. . Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls. , 91-103 (2014).
- Vogel, C., Marcotte, E. M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nature Reviews Genetics. 13 (4), 227-232 (2012).
- Semache, M., Ghislain, J., Zarrouki, B., Tremblay, C., Poitout, V. Pancreatic and duodenal homeobox-1 nuclear localization is regulated by glucose in dispersed rat islets but not in insulin-secreting cell lines. Islets. 6 (4), e982376 (2014).
- Kang, H. S., Beak, J. Y., Kim, Y. S., Herbert, R., Jetten, A. M. Glis3 is associated with primary cilia and Wwtr1/TAZ and implicated in polycystic kidney disease. Molecular and Cellular Biology. 29 (10), 2556-2569 (2009).
- Billova, S., Galanopoulou, A. S., Seidah, N. G., Qiu, X., Kumar, U. Immunohistochemical expression and colocalization of somatostatin, carboxypeptidase-E and prohormone convertases 1 and 2 in rat brain. Neuroscience. 147 (2), 403-418 (2007).
- Westerfield, M. . The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio Rerio). , (2000).
- Nüsslein-Volhard, C., Dahm, R. . Zebrafish: A Practical Approach. , (2002).
- Cheng, C. N., Li, Y., Marra, A. N., Verdun, V., Wingert, R. A. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization. Journal of Visualized Experiments. (89), (2014).
- Brennan, C., Mangoli, M., Dyer, C. E. F., Ashworth, R. Acetylcholine and calcium signalling regulates muscle fibre formation in the zebrafish embryo. Journal of Cell Science. 118 (22), 5181 (2005).
- Liu, D. W., Westerfield, M. Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture. The Journal of Neuroscience. 12 (5), 1859 (1992).
- Borodinsky, L. N., Spitzer, N. C. Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction. Proceedings of the National Academy of Sciences of the United States of America. 104 (1), 335-340 (2007).
- Brunelli, G., et al. Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America. 102 (24), 8752-8757 (2005).
- Hans, F., Dimitrov, S. Histone H3 phosphorylation and cell division. Oncogene. 20 (24), 3021-3027 (2001).
- Yee, N. S., Pack, M. Zebrafish as a model for pancreatic cancer research. Methods in Molecular Medicine. 103, 273-298 (2005).
- Seth, A., Stemple, D. L., Barroso, I. The emerging use of zebrafish to model metabolic disease. Disease Models & Mechanisms. 6 (5), 1080-1088 (2013).
- Fontana, B. D., Mezzomo, N. J., Kalueff, A. V., Rosemberg, D. B. The developing utility of zebrafish models of neurological and neuropsychiatric disorders: A critical review. Experimental Neurology. 299 (Pt A), 157-171 (2018).
- Richter, K. N., et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. The EMBO journal. 37 (1), 139-159 (2018).
- Elsalini, O. A., Rohr, K. B. Phenylthiourea disrupts thyroid function in developing zebrafish. Development genes and evolution. 212 (12), 593-598 (2003).
- Wang, W. D., Wang, Y., Wen, H. J., Buhler, D. R., Hu, C. H. Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 transcription in zebrafish embryo. Biochemical pharmacology. 68 (1), 63-71 (2004).
- Bohnsack, B. L., Gallina, D., Kahana, A. Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling. PloS one. 6 (8), e22991 (2011).
- Inoue, D., Wittbrodt, J. One for all–a highly efficient and versatile method for fluorescent immunostaining in fish embryos. PLoS One. 6 (5), e19713 (2011).
Cite This Article
Hammond-Weinberger, D. R., ZeRuth, G. T. Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae. J. Vis. Exp. (155), e60575, doi:10.3791/60575 (2020).