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

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。
<ol><li>将鱼转移到充满鱼水的产卵池中, 然后用深色织物包裹产卵池, 或将其置于黑暗的橱柜中。<ol>
<li>黑暗使斑马鱼在安乐死之前至少有1小时的适应, 让视网膜色素上皮接近完全分离。30分钟的暗适应足以消除大多数视网膜色素, 虽然它的碎片可能仍然在感光细胞之间的夹层。</li>
	</ol>
</li>
	<li>在热板上的50毫升烧杯中熔化15毫升的石油果冻, 然后将3毫升的液体绘制成3毫升的滑尖注射器。倒置注射器, 放置在试管架中, 让石油果冻冷却。</li>
	<li>在普通的7厘米 X 2.5 厘米的显微镜幻灯片上制作一个可重复使用的切片腔。<ol>
<li>使用清除指甲油, 画窄线, 以创建一个3厘米 X 2.5 厘米的矩形在幻灯片的中心, 让它干, 然后添加另一层指甲油的线条。</li>
	</ol>
</li>
	<li>在成像过程中, 在盖板上准备成像梯以保持切片。切片将形成阶梯的 "阶梯"。<ol>
<li>对于静态成像或最小溶液流动的喷射实验, 使石油果冻梯子由两个扁平宽平行的石油果冻在18毫米方形玻璃盖板上组成。使用注射器将两个扁平的1厘米长的冷油果冻的涂片涂在盖子滑动上, 0.5 厘米。</li>
	</ol>
</li>
	<li>准备好组织切片器。<ol>
<li>用乙醇清洁双刃刀片, 使其风干。剪成季度用剪刀, 首先纵向成一半然后横跨每刀片。</li>
		<li>将切片腔置于组织切片器的舞台上, 在舞台上水平居中, 并用永久性标记标记长边以对齐。</li>
		<li>将刀片部分加载到组织切片器臂上, 确保刀片位于平面上, 而不接触指甲油, 然后轻轻拧紧刀片装置。通过调整旋钮¼转弯, 降低刀片臂的位置, 然后将一小块滤纸放在成像室的中心并进行测试切割。如果纸张未完全切割, 请重新装载刀片式服务器。</li>
	</ol>
</li>
	<li>使用注射器, 放置一个小点的冷油果冻在10厘米培养皿〜1.5 厘米的中心右侧。用用镊子把油果冻朝上按一个成像梯。使另一个小的石油果冻点〜1厘米从入口边缘的成像室。</li>
	<li>用20g 针装上石油果冻注射器, uncap。把针头放在注射器上, 用它在切片室的中心纵长两个1厘米细长的石油果冻。间隔1厘米的小条。</li>
	<li>在一双闭合的镊子周围紧紧环绕30克钨线的中心 4 cm 段, 制作一个可重用的线眼回路工具。通过将钢丝绳向上或向下滑动, 调整线圈的直径, 直到它比斑马鱼的眼睛稍大一些, 通常为2-3 毫米. 拧线两端, 并用实验室胶带将其固定在6厘米的木棒末端。</li>
	<li>准备好铃声的解决方案。<ol>
<li>解冻50X 补充库存解决方案 (表 1) 并将其添加到 HEPES 缓冲的非碳酸氢钠响铃的解决方案 (表 2) 的实验上午;在锥形离心管或无菌玻璃瓶中, 每10毫升的溶液中稀释200µL 的补充液。对于静态成像实验, 每个实验准备至少30毫升的响铃解决方案。灌注实验所需的振铃量取决于流速和总实验时间。</li>
		<li>检查补充溶液的 ph 值是7.4 使用数字 ph 探针或 ph 纸, 并相应地调整与稀释氢氧化钠或 HCl。</li>
		<li>氧补充在冰上的解决方案, 通过冒泡与100% 氧气气体至少5分钟使用标准的医疗氧气罐和调节器装有软管, 或可选的气体涌泉歧管用于灌注。在夹层显微镜附近的密封锥形离心管或无菌玻璃瓶中贮存含氧铃的溶液;使用此解决方案进行解剖, 成像, 稀释染料或药理剂。</li>
		<li>如果在实验中使用了其他解决方案, 如 Na+-自由响铃的解决方案 (表 3), 请重复步骤 1.9. 1.-1.9. 3。</li>
	</ol>
</li>
	<li>在解剖显微镜附近收集培养皿、镊子、微剪刀和其他工具, 并准备一条鱼水冰浴, 用于斑马鱼的安乐死。</li>
</ol>2. 准备视网膜切片 (参见图 1)
<ol><li>在红色环境光下工作, 以尽量减少光的适应 (这可以使视网膜色素粘着更紧密地对眼底), 弄死斑马鱼浸泡在冰浴, 直到触摸反应丢失, 通常1-2 分钟. 把鱼转移到培养皿上, 用手术刀cervically 脱臼, 但不斩首鱼。</li>
	<li>用钢丝圈松开一只眼睛周围的结缔组织, 然后用循环轻轻地将眼睛向前拉一只手。另一方面, 用微剪刀剪下眼睛下面的白色视神经, 小心不要剪掉眼睛的背部。</li>
	<li>用镊子将眼睛转移到冰上冷铃溶液的培养皿中, 然后重复第二眼。保持眼睛在黑暗中或在红灯下, 直到视网膜色素去除。</li>
	<li>在一个低功率解剖显微镜下解剖眼罩在一滴冷铃的解决方案上, 在一个普通的玻璃幻灯片在培养皿。<ol>
<li>用细钳刺穿角膜, 然后用镊子或剪刀轻轻地取出清晰的、脆性的角膜和银色巩膜片。取出并丢弃透镜和大部分巩膜 (请参见图 1A), 并最小地处理隔离的眼罩。</li>
		<li>部分脂肪, 小的巩膜, 和黑色的视网膜色素可以保持连接到眼罩和从视网膜上删除后。如果视网膜在步骤2.4.1 中与 RPE 分离, 则采取同样的步骤, 使用格外小心, 不要损害微妙的孤立视网膜。</li>
		<li>位置眼罩打开侧 (RPE 向上) 在幻灯片上, 并削减到三或季度与一个新的单刃刀片在一个议案 (请参见图 1B)。丢弃高度弯曲的组织。</li>
	</ol></li>
	<li>滤纸上的扁平的视网膜。<ol>
<li>用铃声的溶液弄湿一张滤纸, 放在眼罩片旁边的滑梯上。使用平钳处理完好的湿滤纸时, 避免穿刺。添加冷铃的解决方案, 以覆盖过滤纸和组织。</li>
		<li>用镊子在每只手, 小心地拖动过滤纸在每个眼罩片断之下与 RPE 和感光器面对,即与眼睛的后面朝向。将眼罩件放在过滤纸中心的一行中 (请参见图 1C)。用细钳轻轻地处理眼罩件, 靠近边缘或拐角处。</li>
		<li>为了帮助视网膜粘附滤纸, 请将湿滤纸放在干纸巾上3秒, 使水分向下, 但不要让组织变得干燥。重复直到眼罩片平放在滤纸上。将柔和的吸力应用到滤纸的底部有助于使视网膜扁平化, 但这一步并不重要。</li>
	</ol></li>
	<li>如果眼罩片上有黑色的视网膜色素片, 使用细镊子从一个角落开始轻轻剥开 (参见图 1D), 而组织正坐在一滴铃声的解决方案中。如果视网膜从滤纸上提起, 重复2.5.3 中的吸毛步骤。由于未漂白的视觉色素, 视网膜可能出现粉红色。</li>
	<li>重复视网膜解剖和扁平安装步骤在 2.4-2.6 的第二眼, 如果需要, 保持第一个平面安装的视网膜沉浸在冷铃的解决方案。为了简化切片过程, 两个视网膜的一块可以放在一张滤纸上。一旦视网膜色素去除, 该协议可以在正常的房间光线下进行, 除非实验需要黑暗。</li>
	<li>将滤纸放在幻灯片上, 并将其修剪成一个带有刀片刀片的矩形, 在视网膜线两侧留下0.5 厘米的滤纸。将滤纸移到准备好的切片室, 用镊子将长滤纸边缘推入薄的石油果冻线, 并将视网膜浸入3-4 滴冷铃溶液中。 注: 某些染料, 如亲脂染料, 最好在切片前的这个阶段装入扁平支架。这些可以加载, 邪恶的组织, 并清洗在切片室。例如, C12 558/568 BODIPY 强烈地染色视网膜细胞膜和感光外段 (参见图 4A), 当在室温下 (通常为23-27 摄氏度) 时, 在5µg/mL 上加载15分钟, 然后在多余的响铃溶液中进行清洗。.</li>
	<li>将切片腔体转移到组织切片器阶段, 沿标记线定位长边, 并用实验室胶带将室端固定在舞台上。从一端开始, 在切片臂上用牢固的、柔和的压力切开视网膜和滤纸。检查第一切片是否已完全切割, 然后使用千分尺切割400µm 切片。</li>
	<li>组装成像梯。<ol>
<li>将切片室与成像梯相邻的步骤1.7 中的培养皿中的切片视网膜切片 (请参见图 1E)。用冷铃的溶液填满盘子, 淹没它的内容。</li>
		<li>使用镊子和保持切片淹没, 轻轻地将滤纸和视网膜带到梯子上, 将培养皿从左向右滑动。注意不要直接接触视网膜。旋转切片 90°, 并将过滤纸边缘埋在石油果冻中。</li>
		<li>在梯子上用镊子精细地放置视网膜切片, 以便在解剖显微镜下清晰可见视网膜层 (参见图 1G)。对于梯子的每一端切片, 确保组织正向内向其他切片, 以尽量减少在注射或流动过程中组织的运动。丢弃未粘附在过滤器纸上的任何视网膜切片 (请参见图 2A)。</li>
	</ol></li>
	<li>如果需要, 在这个阶段加载染料进入视网膜切片, 并在成像前在多余的响铃的解决方案中清洗。 注: 碘碘化物 (PI) 和赫斯特33342在室温下分别以5µg/毫升的视网膜切片为20分钟, 对死亡和所有细胞 (参见图 2B) 进行了强力染色。四甲基罗丹明 (TMRM) 在室温下以 1 nM 30 分钟的时间在视网膜内活跃地 respiring 线粒体。</li>
	<li>切片染色时, 准备成像室和注射装置。使用注射器冲洗油管与响铃的解决方案和清除气泡, 连接的开放端的油管到成像室入口, 并关闭旋塞阀。取出注射器并装入注射剂, 然后将其重新连接到油管。</li>
	<li>使用镊子将盖玻片与视网膜切片转移到成像室, 将盖玻片压入成像腔入口边缘附近的石油果冻点 (参见图 1H)。用响铃的溶液填充成像腔以覆盖切片。</li>
</ol>3. 视网膜成像切片
<ol><li>将填充成像室与连接的注射装置放置在直立共焦显微镜的舞台上, 配备20或40X 水浸镜透镜。使用舞台剪辑保护成像室。</li>
	<li>将浸渍透镜放在梯子上, 将焦点放在梯子一端的薄片上, 在昏暗的反光线照射下。检查每个切片的横向方向, 存在的感光外段, 并确保粘附到过滤纸。</li>
	<li>选择最佳的时间推移成像切片, 并配置用于时间推移图像采集的显微镜软件。表 4概述了各种荧光标记和染料的典型成像设置。 注: 图像采集的速率将因显微镜、荧光标记和生物过程而异。例如, 使用800x800 像素分辨率, 2 µs/像素扫描速度, 和 10 s 帧率的成像钙动力学在感光细胞与 GCaMP3 和红色染料。<ol>
<li>如果可用, 请使用该软件跟踪切片中的物理地标 (感光外段、细胞体、原子核), 以帮助在时间失效时进行潜在的重新定位。还可以设置软件来监视切片上的实时荧光。</li>
	</ol>
</li>
	<li>开始成像和监测基线荧光。当荧光穿过切片稳定 (通常在2-5 分钟以内), 继续进行实验。对于注射, 打开注射器旋塞阀, 然后慢慢地压低和拉回活塞两次, 以帮助混合。 注意: 例如, 通过注射 protonophore CCCP 的浓缩溶液来消除线粒体功能, 以达到1µM 在成像室的最终浓度。这会诱发 GCaMP 报告的感光细胞细胞质的健壮 Ca2 +爆裂, 然后 TMRM 报告的线粒体膜电位稳步下降。</li>
	<li>在实验中密切监控切片的漂移, 并利用该软件根据物理标志进行微观调整。通常在注射或灌注过程中的 Z 方向漂移是 < 5 µm。</li>
</ol>4. 在灌注实验中, 当溶液不断改变或流动时, 成像视网膜切片
注: 在灌注实验中, 当溶液被改变或连续流动时, 成像视网膜切片与静态成像或注射实验的设置类似, 并进行以下修改。
<ol><li>而不是步骤1.4 中描述的石油果冻梯子, 使用更坚固的蜡梯来保持视网膜片稳定在溶液流动。<ol>
<li>将两个小平行圆筒未加香料牙蜡0.5 厘米分开在一个盖玻片。在平坦的表面上, 用拇指将每个气缸压向盖玻片的平行边缘。用一个 #1 盖玻片水平地分平两个圆筒 (参见图 1E, 右侧) 然后用刮刀在蜡条之间涂抹一层薄薄的石油果冻。</li>
		<li>在步骤2.10.2 中装配成像梯时, 用细钳将切片的过滤纸边缘按在蜡分数中 (图 1G)。</li>
	</ol>
</li>
	<li>在步骤2.12 中设置灌注, 用 preoxygenated 溶液填充注射器储层, 或使用可选气歧管在每个水库氧溶液。冲洗所有油管与响铃的解决方案, 确保所有的线流动时, 打开和清除大气泡。</li>
	<li>在步骤2.13 中填充成像腔之前, 使用注射器将另一点石油果冻放在盖玻片的每个外露角上, 以防止其在流动过程中横向滑动。</li>
	<li>当成像室填充并安装在显微镜阶段, 打开吸引和连接吸油管。测试响铃的解决方案的流程, 并使用 micropositioner 将吸引管置于流出室, 以便在燃烧室溢出之前抽出少量液体 (通常为2毫升/分钟的溶液表面上约1毫米)。在步骤3.4 中选择切片时保持响铃的解决方案畅通。</li>
	<li>在步骤3.4 中进行实验以进行灌注, 通过在打开第二个解决方案的同时关闭第一个解决方案的旋塞阀来切换解决方案的流程。<ol>
<li>例如, 要从成像腔中耗尽胞外 Na+ , 请使用两个用响铃解决方案或 Na+免费解决方案填充的注射器库 (表 3)。流铃声的解决方案, 建立基线, 然后切换到 Na+-免费解决方案。这将导致较大的胞浆 Ca2 +在感光外段和单元格体中增加 (请参见图 4)。</li>
	</ol>
</li>
	<li>对于重力式灌注系统, 监测每个油藏的溶液水平, 以便在成像过程中流速恒定。顶出水库的需要与含氧溶液, 或保持持续的氧气冒泡与气歧管。</li>
</ol>

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作者声明他们没有竞争的财政利益。

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?...

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。

<table><tbody><tr><td>zebrafish</td><td>Univeristy of Washington South Lake Union Aquatics Facility</td><td></td><td>stocks maintained in-house as stable transgenic lines</td></tr><tr><td>petroleum jelly</td><td>Fisher Scientific</td><td>19-090-843</td><td>for petroleum jelly syringe</td></tr><tr><td>3-mL slip tip syringe</td><td>Fisher Scientific</td><td>14-823-436</td><td>for petroleum jelly syringe</td></tr><tr><td>20g 3.8 cm slip tip needle</td><td>Fisher Scientific</td><td>14-826-5B</td><td>for petroleum jelly syringe</td></tr><tr><td>plain 7 cm X 2.5 cm microscope slide</td><td>Fisher Scientific</td><td>12-550-A3</td><td>for eyecup dissection, slicing chamber</td></tr><tr><td>Seche Vite clear nail polish</td><td>Amazon</td><td>B00150LT40</td><td>for slicing chamber</td></tr><tr><td>18 mm X 18 mm #1 glass coverslips</td><td>Fisher Scientific</td><td>12-542A</td><td>for imaging ladders</td></tr><tr><td>unflavored dental wax</td><td>Amazon</td><td>B01K8WNL5A</td><td>for imaging ladders</td></tr><tr><td>double edge razor blades</td><td>Stoelting</td><td>51427</td><td>for tissue slicing</td></tr><tr><td>tissue slicer with digital micrometer</td><td>Stoelting</td><td>51415</td><td>for tissue slicing</td></tr><tr><td>filter paper - white gridded mixed cellulose, 13 mm diameter, 0.45 &amp;micro;m pore size</td><td>EMD Millipore</td><td>HAWG01300</td><td>filter paper for mounting retinas</td></tr><tr><td>10 cm petri dish</td><td>Fisher Scientific</td><td>FB0875712</td><td>for fish euthanasia, dissection, imaging ladder assembly</td></tr><tr><td>15 cm plain-tipped wood applicator stick</td><td>Fisher Scientific</td><td>23-400-112</td><td>for wire eye loop tool</td></tr><tr><td>30g (0.25 mm diameter) tungsten wire</td><td>Fisher Scientific</td><td>AA10408G6</td><td>for wire eye loop tool</td></tr><tr><td>D-glucose</td><td>Sigma Aldrich</td><td>G8270</td><td>component of supplement stock solution</td></tr><tr><td>sodium L-lactate</td><td>Sigma Aldrich</td><td>L7022</td><td>component of supplement stock solution</td></tr><tr><td>sodium pyruvate</td><td>Sigma Aldrich</td><td>P2256</td><td>component of supplement stock solution</td></tr><tr><td>L-glutamine</td><td>Sigma Aldrich</td><td>G3126</td><td>component of supplement stock solution</td></tr><tr><td>&amp;nbsp;L-glutathione, reduced</td><td>Sigma Aldrich</td><td>G4251</td><td>component of supplement stock solution</td></tr><tr><td>L-ascorbic acid</td><td>Sigma Aldrich</td><td>A5960</td><td>component of supplement stock solution</td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td>component of Ringer&#039;s solution</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P9333</td><td>component of Ringer&#039;s solution</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>C3881</td><td>component of Ringer&#039;s solution</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>S8282</td><td>component of Ringer&#039;s solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; &amp;middot; 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>M0250</td><td>component of Ringer&#039;s solution</td></tr><tr><td>HEPES</td><td>Sigma Aldrich</td><td>H3375</td><td>component of Ringer&#039;s solution</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152</td><td>component of Na&lt;sup&gt;+&lt;/sup&gt;-free Ringer&#039;s solution</td></tr><tr><td>6 N HCl</td><td>Fisher Scientific</td><td>02-003-063</td><td>component of Na&lt;sup&gt;+&lt;/sup&gt;-free Ringer&#039;s solution</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>P5655</td><td>component of Na&lt;sup&gt;+&lt;/sup&gt;-free Ringer&#039;s solution</td></tr><tr><td>50 mL conical centrifuge tube</td><td>Denville Scientific</td><td>C1062-P</td><td>container for Ringer&#039;s solution</td></tr><tr><td>Vannas scissors - 8 cm, angled 5 mm blades</td><td>World Precision Instruments</td><td>501790</td><td>micro-scissors for eyecup dissection</td></tr><tr><td>Swiss tweezers - #5, 11 cm, straight, 0.06 X 0.07 mm tips</td><td>World Precision Instruments</td><td>504510</td><td>fine forceps for eyecup dissection and slice manipulation</td></tr><tr><td>single edge razor blades</td><td>Fisher Scientific</td><td>12-640</td><td>for eyecup dissection and trimming filter paper</td></tr><tr><td>EMD Millipore filter forceps</td><td>Fisher Scientific</td><td>XX6200006P</td><td>flat forceps for handling wet filter paper</td></tr><tr><td>C&lt;sup&gt;12&lt;/sup&gt; 558/568 BODIPY</td><td>Fisher Scientific</td><td>D3835</td><td>stains live cell nuclei; incubate 5 &amp;micro;g/mL for 15 min at room temperature</td></tr><tr><td>propidium iodide (PI)</td><td>Fisher Scientific</td><td>P3566</td><td>stains dead cell nuclei; incubate 5 &amp;micro;g/mL for 20 min at room temperature</td></tr><tr><td>Hoechst 33342</td><td>Fisher Scientific</td><td>62249</td><td>stains live cell nuclei; incubate 5 &amp;micro;g/mL for 20 min at room temperature</td></tr><tr><td>Tetramethylrhodamine, methyl ester (TMRM)</td><td>Fisher Scientific</td><td>T668</td><td>stains functional, negatively-charged mitochondria; incubate 1 nM for 30 min at room temperature</td></tr><tr><td>tissue perfusion chamber</td><td>Cell MicroControls</td><td>BT-1-18/BT-1-18BV [-SY]</td><td>imaging chamber for injection or perfusion</td></tr><tr><td>2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (NBDG)</td><td>Fisher Scientific</td><td>N13195</td><td>fluorescent glucose analog adminitered orally to zebrafish 30 min prior to euthanasia</td></tr><tr><td>Olympus laser scanning confocal microscope</td><td>Olympus</td><td>FV1000</td><td>confocal microscope for visualizing fluorescence of slices at single-cell resolution</td></tr><tr><td>Carbonyl cyanide 3-chlorophenylhydrazone (CCCP)</td><td>Sigma Aldrich</td><td>C2759</td><td>experimental reagent which ablates mitochondrial respiration; treat slices to a final concentration of 1 &amp;micro;M</td></tr><tr><td>miniature aspirator positioner</td><td>Cell MicroControls</td><td>FL-1</td><td>for perfusion</td></tr><tr><td>perfusion manifold, gas bubbler manifold, flow valve, 60cc syringe holder</td><td>Warner Instruments</td><td>various</td><td>for perfusion</td></tr></tbody></table>

preparing fresh retinal slices from adult zebrafish for ex vivo imaging experiments

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。

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...

Watch this Scientific Journal Video about Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments at JoVE.com

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

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。

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

preparing-fresh-retinal-slices-from-adult-zebrafish-for-ex-vivo

Research

JoVE Journal

Neuroscience

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

9.1K 次观看.  University of Washington. 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。

视频: Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

科研

发育生物学

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

神经科学

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

生物学

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼：可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

活斑马鱼幼虫的眼睛去除以检查视觉系统的神经支配依赖性生长和发育

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

通过鱼眼研究糖尿病: 成人 tg (fli: EGFP) 斑马鱼视网膜血管的显微解剖、可视化和分析

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

医学

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

神经元发育过程中的 ROS 活细胞成像

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...