从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验
Summary
影像视网膜组织可以提供无法从传统生化方法收集的单细胞信息。该协议描述了从斑马鱼的视网膜切片的制备共焦成像。荧光基因编码的传感器或指示染料允许可视化许多生物学过程中的不同的视网膜细胞类型。
Abstract
视网膜是一种复杂的组织, 它能启动和整合第一步的视觉。视网膜细胞功能障碍是许多致盲疾病的标志, 未来的治疗取决于对不同视网膜细胞正常功能的基本理解。由于在视网膜细胞环境中, 特定细胞类型的贡献减少, 因此使用生化方法获得这种信息是困难的。由于基因编码的荧光生物传感器数量越来越多, 活体视网膜成像可以提供一个亚细胞水平上许多生物学过程的观点。然而, 这项技术迄今仅限于蝌蚪和斑马鱼幼虫、离体视网膜的最外层眼底层, 或者活动物视网膜的低分辨率成像。在这里, 我们提出了一个方法, 以产生活的前活体视网膜切片从成年斑马鱼的实况成像通过共焦显微镜。这项准备产生横向切片与所有视网膜层和大多数细胞类型可见, 以执行共聚焦成像实验, 使用灌注。转基因斑马鱼表达荧光蛋白或生物传感器在特定的视网膜细胞类型或器被用来从完整的视网膜提取单细胞信息。此外, 视网膜切片可以加载荧光指示器染料, 增加了该方法的通用性。本协议是为斑马鱼锥感光细胞内的成像 Ca2 +开发的, 但有适当的标记, 它可以用来测量在米勒电池、双极性和水平细胞、小胶质、无长突细胞或视网膜上的ca 2 + 或代谢物。神经节细胞。视网膜色素上皮从切片中移除, 因此该方法不适用于研究该细胞类型。通过实践, 可以从一种动物中生成连续切片, 进行多项实验。这种适应性技术为回答许多关于视网膜细胞生物学、Ca2 +和能量稳态的问题提供了有力的工具。
Introduction
斑马鱼 (斑马斑马) 由于其体积小、快速发育和脊椎动物器官系统, 已广泛用于医疗和基础科学研究1。斑马鱼幼虫的天然透明结合了转基因的既定方法, 使活体动物的细胞过程得到了详细的可视化。一些基因编码的荧光生物传感器已瞄准特定的斑马鱼细胞, 以检测 Ca2 + 2, 过氧化氢3, 凋亡激活4和 ATP5。
在体内成像的斑马鱼幼虫导致了神经科学领域的突破性进展, 包括大脑电路6的映射和中枢神经系统疾病的药物开发7。斑马鱼非常适合视觉研究, 因为它们的视网膜具有较高脊椎动物的层流结构和神经元类型, 并且它们显示健壮的视觉行为8,9。在斑马鱼10,11中成功地模拟了几种与人类疾病类似的视网膜退化, 包括在视网膜2内的单个感光细胞的活体成像. 12。
虽然体内斑马鱼幼虫成像是一个有价值的工具, 但随着鱼的生长和色素沉着的发展, 一些药理治疗不能渗透到整个动物身上, 它变得更具挑战性。此外, 某些细胞过程随着发育和年龄的变化而改变, 使以后的时间对理解功能和成人动物疾病的进展至关重要。生物化学方法, 如应用免疫印迹, 定量分析 PCR,O 2 消耗量和 metabolomic 分析可以提供有关视网膜整体生物学的重要线索, 但很难辨别个别细胞类型的贡献受疾病。影像分离的视网膜组织前体绕过这些问题, 而成像扁平的视网膜提供了外部视网膜13的看法, 更深的内视网膜特征被遮蔽。横向视网膜切片, 如在固定免疫组化分析中提出的, 使所有层和细胞类型的清晰的观点, 但只提供一个单一的快照的动态过程涉及正常功能和疾病。
在这里, 我们提出了一个方法来生成体外横向视网膜切片从成年斑马鱼进行成像. 它类似于制备两栖动物和斑马鱼视网膜切片的电生理和形态学研究的方法14,15, 对时间推移成像前体使用共焦的重要修改显微镜。用共聚焦显微镜实时监测生物传感器或染料在切片中的荧光反应, 同时用灌注法提供药理剂。虽然这种方法是为成像感光细胞开发的, 它可能是可行的, 以可视化的米勒电池, 双极细胞, 水平细胞, 无长突细胞, 或视网膜神经节细胞与适当的荧光标记。此外, 切片可以加载荧光细胞渗透染料报告细胞活力, 水泡运输, 线粒体功能, 或氧化还原状态。这种多才多艺的准备允许在整个视网膜上可视化多种亚细胞过程, 包括 Ca2 +动力学, 信号转导和代谢状态。
Protocol
所有动物实验都是由华盛顿大学动物保育和使用委员会批准的。 1. 准备动物和设备 注: 视网膜色素上皮 (RPE) 是一种深色的组织周围的视网膜外, 其色素沉着可以掩盖视网膜特征, 并损害组织时共聚焦成像前体。在黑暗中, 斑马鱼的视网膜色素从视网膜中缩回;深色适应的鱼, 以方便今后去除视网膜色素切片和成像。 将鱼转移到充满鱼水的产卵?…
Representative Results
切片的稳定定位和横向定位是药理学药物注射或灌注成像成功的关键。在共聚焦成像之前仔细检查和重定位切片, 以确保所有视网膜层可见 (图 2A, 切片 ii)。如果切片稍微向前旋转 (图 2A、切片 iii), 则外部段束将可见, 并且可以用镊子进行小调整, 以使所需的视网膜层成为焦点。不应将切片粘附在过滤纸上 (图 2A</s…
Discussion
前体成像的新鲜斑马鱼视网膜切片已被证明是一个多才多艺的工具, 研究感光细胞生物学20,21,22, 是独一无二的, 因为它能够在一个成熟的, 充分的分析单个细胞分化视网膜。通过实践, 有可能进行多项实验的组织从一条鱼, 甚至使用序列切片从同一部分的视网膜。除了对制备用于电生理学研究的两栖类视网膜切片的挑战和?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢拉尔夫尼尔森和丹尼尔. Possin 在制定这项协议时提供了周到的指导, 伊娃 Ma、阿什利. 乔治和盖尔. 斯坦顿为新一代稳定的转基因斑马鱼线。这项工作得到了 NSF GRFP 2013158531 M.G.、NIH 内5T32EY007031 到卫生间和 M.G. 的支持, EY026020 兰伯特和迪萨纳亚克
Materials
| zebrafish | Univeristy of Washington South Lake Union Aquatics Facility | stocks maintained in-house as stable transgenic lines | |
| petroleum jelly | Fisher Scientific | 19-090-843 | for petroleum jelly syringe |
| 3-mL slip tip syringe | Fisher Scientific | 14-823-436 | for petroleum jelly syringe |
| 20g 3.8 cm slip tip needle | Fisher Scientific | 14-826-5B | for petroleum jelly syringe |
| plain 7 cm X 2.5 cm microscope slide | Fisher Scientific | 12-550-A3 | for eyecup dissection, slicing chamber |
| Seche Vite clear nail polish | Amazon | B00150LT40 | for slicing chamber |
| 18 mm X 18 mm #1 glass coverslips | Fisher Scientific | 12-542A | for imaging ladders |
| unflavored dental wax | Amazon | B01K8WNL5A | for imaging ladders |
| double edge razor blades | Stoelting | 51427 | for tissue slicing |
| tissue slicer with digital micrometer | Stoelting | 51415 | for tissue slicing |
| filter paper – white gridded mixed cellulose, 13 mm diameter, 0.45 µm pore size | EMD Millipore | HAWG01300 | filter paper for mounting retinas |
| 10 cm petri dish | Fisher Scientific | FB0875712 | for fish euthanasia, dissection, imaging ladder assembly |
| 15 cm plain-tipped wood applicator stick | Fisher Scientific | 23-400-112 | for wire eye loop tool |
| 30g (0.25 mm diameter) tungsten wire | Fisher Scientific | AA10408G6 | for wire eye loop tool |
| D-glucose | Sigma Aldrich | G8270 | component of supplement stock solution |
| sodium L-lactate | Sigma Aldrich | L7022 | component of supplement stock solution |
| sodium pyruvate | Sigma Aldrich | P2256 | component of supplement stock solution |
| L-glutamine | Sigma Aldrich | G3126 | component of supplement stock solution |
| L-glutathione, reduced | Sigma Aldrich | G4251 | component of supplement stock solution |
| L-ascorbic acid | Sigma Aldrich | A5960 | component of supplement stock solution |
| NaCl | Sigma Aldrich | S7653 | component of Ringer's solution |
| KCl | Sigma Aldrich | P9333 | component of Ringer's solution |
| CaCl2 · 2H2O | Sigma Aldrich | C3881 | component of Ringer's solution |
| NaH2PO4 | Sigma Aldrich | S8282 | component of Ringer's solution |
| MgCl2 · 6H2O | Sigma Aldrich | M0250 | component of Ringer's solution |
| HEPES | Sigma Aldrich | H3375 | component of Ringer's solution |
| Tris base | Fisher Scientific | BP152 | component of Na+-free Ringer's solution |
| 6 N HCl | Fisher Scientific | 02-003-063 | component of Na+-free Ringer's solution |
| KH2PO4 | Sigma Aldrich | P5655 | component of Na+-free Ringer's solution |
| 50 mL conical centrifuge tube | Denville Scientific | C1062-P | container for Ringer's solution |
| Vannas scissors – 8 cm, angled 5 mm blades | World Precision Instruments | 501790 | micro-scissors for eyecup dissection |
| Swiss tweezers – #5, 11 cm, straight, 0.06 X 0.07 mm tips | World Precision Instruments | 504510 | fine forceps for eyecup dissection and slice manipulation |
| single edge razor blades | Fisher Scientific | 12-640 | for eyecup dissection and trimming filter paper |
| EMD Millipore filter forceps | Fisher Scientific | XX6200006P | flat forceps for handling wet filter paper |
| C12 558/568 BODIPY | Fisher Scientific | D3835 | stains live cell nuclei; incubate 5 µg/mL for 15 min at room temperature |
| propidium iodide (PI) | Fisher Scientific | P3566 | stains dead cell nuclei; incubate 5 µg/mL for 20 min at room temperature |
| Hoechst 33342 | Fisher Scientific | 62249 | stains live cell nuclei; incubate 5 µg/mL for 20 min at room temperature |
| Tetramethylrhodamine, methyl ester (TMRM) | Fisher Scientific | T668 | stains functional, negatively-charged mitochondria; incubate 1 nM for 30 min at room temperature |
| tissue perfusion chamber | Cell MicroControls | BT-1-18/BT-1-18BV [-SY] | imaging chamber for injection or perfusion |
| 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (NBDG) | Fisher Scientific | N13195 | fluorescent glucose analog adminitered orally to zebrafish 30 min prior to euthanasia |
| Olympus laser scanning confocal microscope | Olympus | FV1000 | confocal microscope for visualizing fluorescence of slices at single-cell resolution |
| Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) | Sigma Aldrich | C2759 | experimental reagent which ablates mitochondrial respiration; treat slices to a final concentration of 1 µM |
| miniature aspirator positioner | Cell MicroControls | FL-1 | for perfusion |
| perfusion manifold, gas bubbler manifold, flow valve, 60cc syringe holder | Warner Instruments | various | for perfusion |
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Citer Cet Article
Giarmarco, M. M., Cleghorn, W. M., Hurley, J. B., Brockerhoff, S. E. Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments. J. Vis. Exp. (135), e56977, doi:10.3791/56977 (2018).