作者想在斑马鱼国际资源中心承认Katy Murray博士; CCHMC的Stacey Huppert博士和Kari Huppert博士就协议提供了有用的建议;和CCHMC兽医服务，用于养鱼。这项工作得到NIH授权R00AA020514和CCHMC（CY）儿科基因组学中心的研究资助。它也得到了NIH授予P30 DK078392（综合形态核心）消耗性疾病研究核心中心在辛辛那提的支持。

AB WT成年和幼虫斑马鱼根据"实验动物护理和使用指南"（美国国家卫生研究院出版号86-23，1985年修订版）保持在标准条件21 。其使用由辛辛那提儿童医院医疗中心（CCHMC）的机构动物护理和使用委员会批准。 1.准备解决方案<ol><li> 准备蛋水。 <ol><li>通过将40g商业海盐溶解在1L双蒸水（ddH 2 O）中来制备储备盐溶液。搅拌至所有盐完全溶解。 </li><li>通过向100ml ddH 2 O中加入0.1g粉末制备0.1％亚甲基蓝溶液。 小心:如果接触到眼睛或皮肤，亚甲蓝是刺激性的。处理粉末时，请务必戴上实验室外套和手套。 </li><li>合并28.125毫升的储备盐并在50mL锥形管中加入7mL亚甲基蓝溶液。混合好将此混合物加入15 L的ddH 2 O.彻底摇匀并在室温下储存。 </li></ol></li><li>通过向100ml ddH 2 O中加入12.1克Tris粉末，制备100mL的1M Tris-HCl。使用盐酸（HCl）调节pH至9.0。 注意:如果吸入，HCl会引起严重烧伤和粘膜刺激。始终穿实验室外套和手套，并将化学品盖子上的瓶子装起来。 </li><li> 制备0.4％三卡因（3-氨基苯甲酸乙酯甲磺酸乙酯）溶液。 <ol><li>将2克三奈米粉末溶解在489.5毫升的ddH 2 O中。加入10.5毫升Tris-HCl，pH 9溶液。与搅拌棒混合，直到所有粉末溶解。调整至pH 7.0。 小心:三仙碱粉可能是呼吸道刺激物。处理粉末时，请务必戴上防尘面具。 </li><li>将等分试样将45毫升三卡因溶液加入50mL锥形管中。保持solution在4°C即刻使用，-20°C长期储存。 </li></ol></li><li>通过在玻璃介质瓶中混合30 mL 95％乙醇，10mL 37％甲醛，2 mL冰醋酸和58 mL ddH 2 O，制备Dietrich固定剂。通过旋转和储存在RT混合。 注意:甲醛是一种疑似致癌物质，任何甲醛溶液都应用于化学防护罩。冰醋酸会引起严重的皮肤灼伤和眼睛损伤。始终戴实验室外套和手套，并在化学品罩中处理原液。甲醛，冰醋酸和95％乙醇都是易燃液体，应存放在指定的易燃柜内。 注意:Dietrich的固定剂在室温下稳定1年。 </li><li>通过加入80克氯化钠（NaCl），2克氯化钾（KCl），14.4克磷酸二钠（Na 2 HPO 4 ）和2份磷酸盐缓冲盐水（PBS）溶液，制备1升10倍磷酸盐缓冲盐水（PBS）溶液。4g磷酸二氢钾（KH 2 PO 4 ）至800 mL ddH 2 O.使用氢氧化钠（NaOH）调节至pH 7.4，并加入ddH 2 O至1 L终体积。在高压灭菌后，在清洗1分钟并在121℃温育20分钟的液体循环下将该溶液存放在室温下。 </li><li> 通过将100mL的10×PBS溶液稀释到900mL的ddH 2 O中来制备1L的1x PBS。通过摇动并在高压灭菌后在室温下储存。 </li><li>准备3％的琼脂糖在玻璃培养瓶中进行胚胎移植。 <ol><li>将3g琼脂糖加入到100mL ddH 2 O中，通过旋转混合。通过微波加热混合物以溶解琼脂糖，并在加热期间仔细观察以确保沸腾最小。 小心:尽管加热不足，瓶子从微波炉中取出时可能会很热。使用防护手套或手套从微波炉中取出瓶子。 </li><li>从微波炉中取出瓶子。轻轻地旋转，寻找任何不完全溶解的晶体;如果没有晶体，溶液就可以使用了。储存3％琼脂糖在室温下再次加热使用前。 </li></ol></li><li> 过滤Harris苏木精储备溶液以除去沉淀的任何固体。 <ol><li>将咖啡过滤器折叠一半，以使过滤器产生锥体。打开侧面板，使液体通过过滤器，使任何碎屑被捕获。 </li><li>将过滤器放入漏斗中，将漏斗放入玻璃介质瓶中。将苏木精逐渐倒入过滤器中。 注意:苏木精在眼睛接触，摄入和吸入的情况下是有害的。始终戴实验室外套和手套，并在化学品罩中处理原液。 </li></ol></li><li>通过将125μL12N HCl加入到250mL的ddH 2 O中来制备0.05％的HCl。 </li><li>通过将25mL的1％e组合制备曙红Y-氧化物B溶液混合物osin Y（aq），2.5mL 1％的福昔宁B（aq），195mL的95％乙醇和1mL的冰醋酸在玻璃瓶中。通过旋转和储存在RT混合 注意:曙红Y在眼睛接触，摄入和吸入的情况下是有害的。始终戴实验室外套和手套，并在化学品罩中处理原液。 </li><li>通过向98mL蛋水中加入2mL纯乙醇制备2％乙醇。 小心:乙醇是眼睛刺激性的。处理乙醇时，请务必戴上适当的保护装置。它也是易燃的，应该相应地处理和存储。 注意:在进行急性乙醇治疗之前，每次准备新鲜的2％乙醇。 </li></ol> 2.在斑马鱼幼虫中进行急性乙醇处理<ol><li> 为了产生胚胎，每个交配罐用一个野生型雄性和一只WT雌性鱼形成十字架。通过在配合槽中插入塑料分隔器来分开它们。 注意:设置在一天的最后喂养之后的十字架，以便鱼很好地喂养。 <ol><li>第二天早上8点，拉分隔线，让对配合。 </li><li> 1小时后，将成年鱼返回原来的坦克。通过将含有胚胎的水倒入过滤器来收集胚胎。在100毫米培养皿中过滤器过滤，并使用含有蛋水的洗涤瓶将胚胎洗涤到培养皿中。将胚胎放在28℃的培养箱中。 </li><li>当胚胎达到至少4个细胞阶段（1 hpf）时，用巴斯德吸管器去除水中未受精的胚胎和碎屑，并将受精胚胎的培养皿放置在28℃的培养箱中。将培养箱中的胚胎保持在28℃，直到受精后96小时。 </li><li>通过以1-10（体积/体积）比将三烯酮溶液加入到蛋水中来麻醉幼鱼。 </li><li>选择最多四十个96 hpf的幼虫，使用充气的膀胱巴斯德吸管，并将它们均匀分成两种新的培养皿。 </li><li>取出尽可能多的残留蛋水。在一个密度为1幼虫/ mL的情况下，将含有2％乙醇的蛋水加入到一个培养皿中。将相同数量的蛋水加入另一道菜作为对照。 </li><li>将对照和乙醇处理的幼虫保持在鱼室24小时。 注意:在14小时光/ 10小时黑暗循环的鱼室进行乙醇处理，导致比在孵化器黑暗中进行实验更一致和更健壮的肝损伤。 </li><li>在1.5 mL离心管中收集幼虫，每只管不超过20只幼虫。 </li><li>从幼虫中除去尽可能多的液体，并在化学防护罩中加入至少1 mL（10x组织体积）的Dietrich固定剂。让鱼在室温下固定在营养器上至少24小时。 注意:Dietrich的固定剂在几周内稳定。 </li></ol></li></ol> &lt;p class ="jove_title"&gt; 3。组织盒的制备和加工<ol><li> 设置必需数量的纸盒和两个蓝色活检垫每个盒。在每个盒子的底部放置一个活检垫。用铅笔标记每个盒子，清楚地标识样品。 注意:不要使用笔或标记来标记纸盒，因为标签将在以后的步骤中丢失。 </li><li>将幼虫嵌入3％的琼脂糖中，以确保所有样品的方向一致。 <ol><li>从化学防护罩的管中取出固定剂，并用1 mL 1x PBS将幼虫洗涤3次，每次5 min。在洗涤过程中，将管子放置在恒温器上。 </li><li>在洗涤期间，使用微波将制备的3％琼脂糖加热到水中以熔化固体。一次加热30秒，仔细观察以尽量减少沸腾。将液体琼脂糖保持在温度为90℃的热板上，同时温和搅拌。 </li><li>使用移液器移液高达8升将其植入塑料组织学模具中，尽可能多地移除PBS。使用带有尖端切口的移液管，用3％琼脂糖完全填充模具。 </li><li>使用胰岛素注射器，将幼虫收集到模具的中心并将其推到底部。对于矢状切片，将幼虫定位成一条线，头朝向模具顶部。为了确保肝脏以一致的方式定向，请转动幼体，使身体的左侧面朝下且平直于模具的底部。 注意:因为肝脏位于身体的左侧，所以这样的定位可以最大限度地减少在到达肝脏之前需要切割的部位数量。保持幼虫尽可能接近。 </li><li>将模具放在一边4-5分钟，以使琼脂糖完全固定。从模具中取出琼脂糖块，并使用剃刀刀片来修剪幼虫周围的琼脂糖。将块放在末端，并将其厚度减半最后的块大约2-3毫米厚。 </li><li>将小琼脂糖块转移到制备的组织盒（步骤3.1），并将第二个活检垫放在块的顶部。关闭盒子并将完全组装的盒子放入可密封的容器中，并装入新鲜制备的在ddH2O中的70％乙醇。 注意:如果盒子不能立即进行处理，请将其放置在容器内的70％乙醇中，以避免在4°C的容器内损失固定。纸盒在4℃下稳定长达3天。 </li></ol></li><li> 组织加工 注意:样品可以提交给配备有组织处理器的组织学或病理学实验室，以在石蜡中加工盒。要求标准的O / N处理程序，而不是简短的程序，以便石蜡可以完全穿透琼脂糖。 <ol><li>通过将它们通过稀释系列的乙醇，随着酒精量的增加而转移到组织盒中脱水组织如下:70％乙醇2次，每次45分钟; 80％乙醇，45分钟; 95％乙醇，2次，每次45分钟; 100％乙醇，2次，每次45分钟。 </li><li>用溶剂（100％二甲苯，2次，每次45分钟）更换醇，以使石蜡渗入组织。 注意:二甲苯是刺激性的，如果吸入有害。一定要使用化学防护罩。二甲苯是易燃的，必须相应存放。 </li><li>在60-65°C的培养箱中，以0 / N的比例将盒子孵育。保持盒子温暖直到嵌入。 </li></ol></li><li> 将处理过的琼脂糖块嵌入石蜡。 <ol><li>从包埋机的加热抽屉中取出一个盒子，从盒子中取出盒子的盖子和顶部活检板。用液体石蜡填充组织学模具，并将其保存在嵌入机上的温热板上。 </li><li>使用温暖的镊子，从cassett拿起琼脂糖块e并用石蜡转移到组织学模具中。确保最接近表面的鱼的琼脂糖块侧面朝下。丢弃底部活检垫，并将整个组织学模具转移到嵌入机上的冷却板上。 </li><li>使用镊子，将琼脂糖块快速放置在模具的中心;将块放在模具的底部。一旦块正确放置，将盒子的底部放在组织学模具的顶部，并用液体石蜡填充一半。将模具直接放在4°C板上，不要打扰块至少10-15分钟，使石蜡固化。当第一个块正在设置时，对剩余的块重复此过程。 </li><li>一旦为所有块设置石蜡，通过轻轻按压模具将其松散，从石蜡块中取出组织学模具。这些石蜡块可以无限期地在室温下储存。 </li></ol></li></OL&gt; 石蜡块的切片<ol><li>切片前一天，使用切片机在石蜡块中面对去除覆盖组织的多余石蜡。一段时间为5μm，直到组织在石蜡块表面露出。停止并丢弃所有切割的部分。在4°CO / N下，将1个PBS中的块面朝下浸泡。 注意:块应浸泡至少8小时。在一天结束时开始浸泡。如果块在1x PBS中放置太久，组织会膨胀并变形。 </li><li>一次从1x PBS中移除一个区块，并使用切片机切割5-μm切片。通过使用镊子或刷子轻轻地将最后一部分从叶片上拉出来，将部分带与刀片分开。使用镊子拿起最后一部分的色带，并将其转移到42°C的水浴中。将色带漂浮在水面上至少5分钟。 注意:n转移部分，不要让镊子在与石蜡接触的同时接触水。否则，石蜡将熔化到镊子上，使分离非常困难。如有必要，使用清洁镊子将部分分成更小的部分，以便它们可以轻松地装配到幻灯片上。轻轻地触摸各部分之间的接缝，以创建分离而不会损坏。 </li><li>将一个充电的滑块以45度的角度放入水中，并小心地将其放置在要收集的部分组下面。小心地从水中提起幻灯片，并让部分贴在幻灯片上。 </li><li>使用无绒纸巾从切片中涂抹任何多余的水分。将幻灯片放在幻灯片支架或盒子中。继续对块进行切片，直到收集所需的组织。对所有块重复分段过程。 </li><li>将载玻片在55℃的培养箱或烘箱中烘烤3-16小时以熔化石蜡。从烤箱中取出载玻片并允许它们o开始染色前先冷静。 注意:在烘烤之前和之后，石蜡切片可以无限期地储存在RT上。 </li></ol> 5.石蜡切片的苏木精和伊红染色<ol><li>将载玻片浸入100％二甲苯中15分钟，使其脱蜡;将其更换为新鲜的100％二甲苯另外15分钟。 </li><li>通过将它们浸入下列系列分级乙醇中，使液体干净地从载玻片上运行，从而使载玻片再次水化。对于每种溶液，浸渍8-10次，每次浸渍2次:100％乙醇，100％乙醇，95％乙醇，95％乙醇，70％乙醇，50％乙醇，30％乙醇和去离子水。 注意:该协议可以在这里停止，幻灯片可以在室内在水中放置数小时。如果需要，载玻片可以在4°C储存在水中。 </li><li>将载玻片置于100％过滤的哈里斯苏木精中4分钟。立即用去离子水将其转移回容器。运行去离子水进入t他的后角是最远的部分。定期清空容器，直到水不再变紫。 注意:不要让水直接滑到幻灯片上，因为这些部分可能会从幻灯片中脱落。 </li><li>使用鹅颈灯快速检查解剖显微镜上的苏木精强度;不要让幻灯片干燥。如果污渍达到所需的强度，请继续下一步。如果颜色不够黑，请将载玻片置于100％苏木精中1分钟，然后重新洗净，再次检查。 注意:苏木精需要足够黑，以便在曙红染色期间颜色不会消失。然而，如果在细胞质中观察到苏木精染色，则切片被过度染色。 </li><li>将载玻片在0.05％HCl中浸渍两次，并立即用干净的去离子水将其转移回容器。清空水并用水重新装满容器两次。 </li><li>将幻灯片转移到95％等牛油30秒，然后将其转移到具有95％乙醇的新容器中30秒。 </li><li>将载玻片放入伊红Y-氧氟烷B溶液中2分钟。将载玻片转移到以前的95％乙醇容器中，并快速检查解剖显微镜下的颜色强度。如果染色足够，请继续下一步。如果没有，请返回曙光解决方案30秒，然后重新检查，并根据需要重复。 注意:足够的伊红染色是粉红色的，与苏木精染色不同。在显微镜下检查时，请确保从幻灯片背面擦拭任何溶液，以免观察到假色。因为曙红由95％乙醇组成，所以在95％乙醇中长时间检测颜色强度可能会浸出一些颜色。 </li><li>将载玻片转移至100％异丙醇15秒。用新鲜的100％异丙醇代替，将载玻片放回异丙醇中15秒。重复此p共焙烧6次异丙醇洗涤。 小心:异丙醇是眼睛和呼吸道刺激物。请务必戴上适当的防护装备，避免飞溅。它也是高度易燃的，应该相应地进行存储和处理。 注意:要保存试剂，请仅丢弃第一（最小）异丙醇洗涤液。将其他五种洗涤溶液保存在编号为1至5的瓶中，以重新用于下一次染色。重复洗涤时，从"1"的瓶子开始，使用后丢弃。一旦洗涤"2"，将其倒入瓶子"1"，等等。最后的洗涤应该是新鲜的异丙醇。 </li><li> 将载玻片置于100％二甲苯中3分钟。一次取出一张幻灯片并放置盖玻片。 <ol><li>添加足够的固定介质以覆盖各部分并将其浸入100％二甲苯中。 注意:此步骤将平滑安装介质的表面，并清除任何气泡。 </li><li>应用盖玻片到幻灯片的底部。 注意:安装介质应将滑块拉到位。如果盖玻片不是直立或完全就座，请轻轻地将其插入到位。 </li><li>在纸巾上涂抹任何多余的安装介质，直到看到细线。将纸巾擦拭到二甲苯中，擦拭幻灯片的背面以除去已滴下的任何介质。将幻灯片平放在坚固但移动的表面上，如一块纸板。以相同的方式覆盖所有剩余的幻灯片。让二甲苯在罩中蒸发10分钟。 注意:任何被二甲苯污染的物质都应该被清除，并放在罩中密封的容器中，以防止任何蒸气逸出。 </li></ol></li><li>允许安装介质在RT O / N下硬化。 </li></ol>彩色幻灯片的成像和存储<ol><li>复合倒置显微镜上的图像部分18 。 注意:幻灯片可以保留在室内温度无限期</li></ol>

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作者宣称他们没有竞争的经济利益。

目前的方案描述了斑马鱼幼虫急性乙醇处理的详细程序和随后的H＆E染色组织病理学分析。急性乙醇治疗应不迟于受精后96小时进行，因为这是斑马鱼肝开始表达酒精代谢酶13的阶段 。 2％乙醇是幼虫可耐受的最大剂量13,14 。乙醇处理的幼虫在处理8小时后开始出现肝脂肪变性，发生脂肪变性的幼虫的百分比持续上升，直到24小时连续处理14 。斑马鱼幼虫从蛋黄中仅吸收营养，直至120 hpf。因此，不推荐乙醇处理超过120 hpf，因为禁食也可能导致脂肪变性14 。与其他实验室发布的方案相比实施例 13,24，在现行方案中进行的主要修改是乙醇处理在具有14小时光/ 10小时黑暗周期的鱼类设施中进行。这导致比在培养箱中在黑暗中进行治疗时更一致和更强健的脂肪反应。这可能与以下事实有关:斑马鱼肝脏中的脂质代谢基因显示响应于明暗周期25的每日节奏表达。 ALD是一种慢性疾病，需要多年的酗酒。目前的乙醇处理方案的关键限制是它只会引起酒精对肝脏的急性影响。乙醇浓度高达48小时以上的治疗导致死亡率高，阻止了慢性肝损伤的研究。最近的一项研究发现，连续治疗成人斑马鱼与1％乙醇达三次几个月导致脂肪变性，脂肪性肝炎和纤维化12 。一个有希望的未来方向可能是使用急性乙醇治疗方案来鉴定ALD的潜在调节剂，然后验证其在慢性损伤模型中的作用。 进行H＆E染色以评估急性乙醇治疗引起的肝细胞损伤。由于在斑马鱼中易于进行荧光成像，在斑马鱼肝组织学中不常规使用H＆E染色，其程序不太清楚。目前的方案提供了在幼虫肝脏中H＆E染色的一步一步描述。选择正确的固定剂是H＆E染色的第一个也是最重要的步骤。虽然10％的福尔马林缓冲液和4％的PFA通常用于组织学，但它们都会导致组织收缩和部分肝脏脱落。 10％福尔马林固定导致肝脏中细胞质的损失ocytes。 4％PFA固定导致肝细胞之间的人为间隙。在用固定剂固定的肝脏中，曙红染色似乎比苏木精染色弱得多。基于酸的Dietrich的固定剂更适合于斑马鱼肝脏的H＆E染色，因为它可以保留细胞细胞并减少收缩。它也似乎渗透脂肪组织，如肝脏，比福尔马林和PFA快。苏木精和伊红染色更加平衡。 Dietrich固定剂的一个注意事项是它与基因组DNA提取不相容。在试验实验中，从使用10％福尔马林，4％PFA或Dietrich固定剂26固定的幼虫中提取基因组DNA。将幼虫在50μL50mM NaOH中在95℃下孵育20分钟，然后冷却至4℃。然后加入5μL1M Tris-HCl，pH8.0以中和碱性溶液。经过短暂的离心，上清液w如PCR中所用。使用相同的PCR引物和PCR程序，来自福尔马林和PFA固定的幼虫的基因组DNA产生具有预测大小的PCR产物，而来自Dietrich固定化固定幼虫的基因组DNA未能产生任何PCR产物。 可以在石蜡切片和冷冻切片上进行H＆E染色。然而，石蜡切片比冷冻切片具有以下优点:1）尽管石蜡切片可以无限期地在室温下储存，但冷冻切片只能在-80℃下储存长达一年。 2）对于冷冻切片，细胞内冰晶的形成可能扰乱细胞形态和亚细胞细胞。此外，冷冻切片通常比石蜡切片更厚。与从石蜡切片产生的那些相比，这可能导致组织形态的差的图像。 当肝组织制备石蜡块时，目前的方案将幼虫嵌入琼脂糖。幼虫横向定位，身体的左侧面朝下并且平放在模具的底部。这确保了肝脏的一致定位，使得当切片顺序切割时，肝脏的等同区域可以从鱼类与鱼类进行比较27 。确保成功H＆E染色的另一个关键步骤是色彩发展。至关重要的是要经常检查染色，直到达到所需的颜色强度。 H＆E染色应用于肝损伤的初步评估。乙醇处理的幼虫在肝细胞中显示出圆形液滴的过度沉积，表明脂肪变性13,14,18。使用脂质染料（如油红O和尼罗红）进行标记是必需的，以确认这些液滴确实是脂质。肝血管被治疗的动物中的els出现扩张和肿胀。应进行扫描电子显微镜和透射电子显微镜检查正弦波的超微结构变化。之前已经报道，通过免疫荧光检测，乙醇处理的斑马鱼肝细胞外基质蛋白沉积增加18 。然而，鉴于在处理的鱼18中纤维形成基因的表达水平仅适度增加，H＆E可能不足以检测这种少量的细胞外基质蛋白。在长期受伤的成年斑马鱼肝脏上测试相同的固定和染色方法，并且足以检测纤维化（尹，未发表的数据）。 虽然目前的方案是针对斑马鱼幼虫肝脏组织学检查而定制的，但它对斑马鱼研究界的应用更为广泛，如同same方案可以应用于其他组织和成年斑马鱼。

酒精饮酒引起的酒精性肝病（ALD）是酒精相关发病率和死亡率的主要原因。在美国，近一半的肝脏疾病死亡涉及酒精1 ，ALD负责近1/3的肝脏移植2 。 ALD有广谱。脂肪变性，其特征在于肝细胞中过量的脂质积累，发生在重度饮酒的早期阶段，并且在酒精停止使用时是可逆的。在遗传和环境因素和持续饮酒的影响下，肝脂肪变性可能发展为酒精性肝炎，最终导致肝硬化3 。使用啮齿动物ALD模型的研究为疾病提供了深刻的见解，但它们有局限性（参考文献3 ）。口服酒精饮食只会引起啮齿动物脂肪变性4/ sup&gt; 5 。炎症和纤维化的发展需要第二次侮辱6,7或慢性胃内输注，这是侵入性和技术上具有挑战性的8,9 。硬糖斑马鱼也发生肝损伤，对慢性和急性酒精治疗10,11,12,13,14,15。特别是，幼虫斑马鱼是一种有吸引力的互补模式生物，其中研究急性酒精性肝损伤10,11,13,15。斑马鱼肝功能并在4天内产生乙醇代谢的关键酶（dpf）13,16,17。乙醇可以直接加入到水中，暴露于2％乙醇24小时足以在斑马鱼幼虫13,15中诱导肝脂肪变性和纤维化反应。 据报道，用2％乙醇处理24小时导致斑马鱼幼虫13的组织乙醇浓度为80mM。其他人已经表明幼虫耐受这一浓度，并且在处理的动物中看到的肝脏表型对乙醇暴露具有特异性11,13,15,18。然而，由于80mM在人体中几乎是致命的19 ，重要的是评估乙醇处理的斑马鱼的肝脏组织学和dete对人类的生理相关性。 斑马鱼幼虫的快速外部发育和半透明使得有可能实时和固定样品中肝脏中酒精的作用。细胞型特异性荧光转基因品系的可获得性以及共聚焦显微镜近期的进展有助于研究不同肝细胞类型如何改变其响应于急性乙醇处理的形态和行为11,15。然而，当研究肝组织学时，荧光转基因斑马鱼的共聚焦成像不能完全替代苏木精和曙红（H＆E）染色。使用转基因斑马鱼同时标记所有肝细胞类型需要产生个体转基因品系，每个转基因品系用独特的荧光团标记一个肝细胞类型。将不同的转基因背景引入同一条鱼需要繁殖多代，这是耗时且昂贵的。需要额外的免疫荧光染色来检测细胞外基质成分。另一方面，H＆E染色同时标记所有肝细胞类型和细胞外基质组分，从而提供肝脏的概况20 。此外，它容易显示肝脏疾病的几种组织病理学特征，如肝细胞死亡，脂肪变性和纤维化。虽然H＆E是哺乳动物肝脏组织学中的常规染色体，但它并不常用于斑马鱼肝脏研究，而且协议不太成熟。 这项工作描述了斑马鱼幼虫急性乙醇治疗方案和H＆E染色后续组织学分析。 H＆E染色方案可用于所有肝脏发育和功能研究。此外，石蜡切片可用于免疫组织化学和其他特殊治疗包括肝脏病理学，包括三色染色，网状蛋白染色等 。

<table><tbody><tr><td>1.5 mL centrifuge tubes</td><td>E &amp;amp; K Scientific</td><td>280150</td><td></td></tr><tr><td>15 mL conical tubes</td><td>VWR International</td><td>89039-664</td><td></td></tr><tr><td>50 mL conical tubes</td><td>VWR International</td><td>89039-658</td><td></td></tr><tr><td>95% ethanol (EtOH)</td><td>Decon Labs, Inc.</td><td>2801</td><td>Flammable</td></tr><tr><td>Acetic acid</td><td>Newcomer Supply</td><td>10010A</td><td>Irritant</td></tr><tr><td>Agarose</td><td>Research Products International</td><td>9012-36-6</td><td></td></tr><tr><td>Aluminum jar rack holder</td><td>Newcomer Supply</td><td>5300JRK</td><td></td></tr><tr><td>Bacteriological petri dishes with lid</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>Biopsy pads</td><td>Simport</td><td>M476.1</td><td></td></tr><tr><td>Charged slides</td><td>Fisher Scientific</td><td>12-550-16</td><td></td></tr><tr><td>Clear mounting media</td><td>Fisher Scientific</td><td>8310-16</td><td>Can be substituted with other clear mounting media</td></tr><tr><td>Commercial sea salts</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>Disposable microtome blades</td><td>Fisher Scientific</td><td>4280L</td><td></td></tr><tr><td>Dissecting microscope</td><td>Leica Biosystems</td><td>Leica Mz 95</td><td></td></tr><tr><td>Enclosed tissue processor</td><td>Leica Biosystems</td><td>ASP300 S</td><td></td></tr><tr><td>Eosin-Phloxine stain set</td><td>Newcomer Supply</td><td>1082A</td><td></td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td>Flammable</td></tr><tr><td>Formaldehyde solution, ACS reagent, 37 WT. % in H&lt;sub&gt;2&lt;/sub&gt;0, contains 10 - 15% methanol as stabilizer (to prevent polymerization)</td><td>Sigma-Aldrich</td><td>252549</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Formalin, Buffered, 10%</td><td>Fisher Scientific</td><td>SF100-4</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Graduated media bottle</td><td>VWR International</td><td>16159-520</td><td></td></tr><tr><td>Harris hematoxylin</td><td>Poly Scientific R&amp;amp;D Corp.</td><td>s212</td><td>Irritant</td></tr><tr><td>Histology molds</td><td>Sakura Finetek USA Inc</td><td>4557</td><td></td></tr><tr><td>Hot plate/Stirrer</td><td>VWR International</td><td>47751-148</td><td></td></tr><tr><td>Hydrochloric acid (HCl)</td><td>Fisher Scientific</td><td>A144</td><td>Irritant</td></tr><tr><td>Incubator</td><td>VWR International</td><td>97058-220</td><td></td></tr><tr><td>Insulin syringes</td><td>BD Medical</td><td>BD-309301</td><td></td></tr><tr><td>Inverted compound microscope</td><td>Carl Zeiss Microscopy</td><td>491912-9850-000</td><td></td></tr><tr><td>Isopropanol</td><td>Newcomer Supply</td><td>12094E</td><td>Flammable</td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140</td><td>Irritant</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>Leica Jung BioCut 2035&amp;nbsp;</td><td></td></tr><tr><td>Nutating mixer</td><td>VWR International</td><td>82007-202</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148-1KG</td><td>A suspected carcinogen; irritant</td></tr><tr><td>Pasteur pipet</td><td>VWR International</td><td>53283-916</td><td></td></tr><tr><td>Pipette pump (10 mL)</td><td>VWR International</td><td>53502-233</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Potassium phosphate, monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td></td></tr><tr><td>Razor blades</td><td>Grainger</td><td>4A807</td><td></td></tr><tr><td>Slide Staining Kit</td><td>Newcomer Supply</td><td>5300KIT</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Fisher BioReagents</td><td>S318-500</td><td>Very hazardous</td></tr><tr><td>Sodium phosphate, dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S3264</td><td></td></tr><tr><td>Stainless steel strainer (5 inch diameter)</td><td>Adaptive Science Tools</td><td>L0906045in</td><td></td></tr><tr><td>Tissue cassettes</td><td>Simport</td><td>M505.12</td><td></td></tr><tr><td>Tissue embedding center</td><td>Sakura Finetek USA Inc</td><td>#5100</td><td></td></tr><tr><td>Tissue wipers, 1-Ply</td><td>Fisher Scientific</td><td>06666A</td><td></td></tr><tr><td>Transfer pipets</td><td>Fisher Scientific</td><td>137117M</td><td></td></tr><tr><td>Tricaine powder/Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma-Aldrich</td><td>A5040</td><td>Irritant</td></tr><tr><td>Tris base, primary standard and buffer</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Wash bottle, low-density polyethylene, wide mouth</td><td>Nalge Nunc International</td><td>2402-0750</td><td></td></tr><tr><td>Xylenes</td><td>Fisher Scientific</td><td>X3S-4</td><td>Irritant</td></tr></tbody></table>

histological analyses of acute alcoholic liver injury in zebrafish

酒精性肝病（ALD）是指由于急性或慢性酒精滥用而导致的肝损害。这是酒精相关发病率和死亡率的主要原因之一，影响了美国200多万人。更好地了解酒精诱导的肝损伤的细胞和分子机制对于开发ALD的有效治疗至关重要。斑马鱼幼虫在接触2％乙醇仅24小时后出现肝脂肪变性和纤维发生，使其可用于急性酒精性肝损伤的研究。这项工作描述了斑马鱼幼虫急性乙醇治疗的程序，并表明它引起肝血管的脂肪变性和肿胀。还描述了针对斑马鱼幼虫肝组织学分析优化的苏木精和曙红（H＆E）染色的详细方案。 H＆E染色比免疫荧光具有几个独特的优点，因为它标志着所有的生物er细胞和细胞外组分同时并且可以容易地检测肝损伤，例如脂肪变性和纤维化。鉴于斑马鱼在建模毒素和病毒诱导的肝损伤以及遗传性肝脏疾病方面的用量越来越多，该协议作为所有这些研究中进行的组织学分析的参考。

10％缓冲福尔马林和4％多聚甲醛（PFA）是用于组织学实践的两种最常见的固定剂。然而，他们没有为斑马鱼肝组织提供最佳的固定结果（ 图1和表1 ）。用10％福尔马林或4％PFA固定经常导致收缩，在肝脏和周围组织之间产生大的间隙（ 图1A ， B ; 图1B提供组织收缩的实例）。肝组织的一些部分也可能脱离该部分。肝细胞的细胞质经常丢失（ 图1A ， 1B ）。两种固定剂都会导致肝细胞变形，因为它们收缩或膨胀，并且不再保持上皮细胞特征的柱状形态。 PFA固定的另外一个问题是在肝细胞之间存在不是真实的，导致组织看起来骨折的空间（ 图1B ）。此外，当使用这两种固定剂中的任一种时，肝细胞用苏木精染色良好，但与曙红相似（ 图1A ）。酸性Dietrich的固定剂克服了福尔马林和PFA固定剂的问题（ 图1C ）。 在人类ALD中，由小和大液滴脂肪组成的脂肪变性在中心静脉附近最突出，并且随着严重程度的增加向外延伸到门静脉三联征22 。在斑马鱼中，用96-120hpf的2％乙醇处理也诱导肝脂肪变性，这与肝细胞中圆滴的过度沉积有关（ 图2B ，箭头）。但是，液滴不会显示ta类似于人类ALD中的分布模式，因为斑马鱼肝门静脉和中央静脉没有明确区别23 。与对照肝脏相比，乙醇处理的幼虫肝脏中的肝血管肿胀（ 图2B ，星号）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55630/55630fig1.jpg" /> 图1 : 在120 hpf下用差异固定剂固定的斑马鱼幼虫肝脏中H＆E染色的比较。 （ A ）10％福尔马林，RT O / N; （ B ）4°CO / N时的4％PFA; （ C ）Dietrich在室温下固定24 h。使用10％的福尔马林，肝细胞经常失去其细胞质（A）。组织没有用曙红正确染色，呈紫色。修复后4％PFA，肝细胞（B）之间存在差距。 4％PFA导致肝组织收缩，所以肝脏和周围组织之间似乎存在较大的间隙。 B中的虚线标记周围组织的边界。 Dietrich的固定剂在三（C）中提供了最佳的结果。刻度棒=20μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55630/55630fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55630/55630fig2.jpg" /> 图2 : 急性乙醇治疗导致斑马鱼幼鱼肝脏脂肪变性和血管肿胀。 （ A ）在对照组，未处理的WT幼虫中肝脏的H＆E染色。 （ B ）用2％乙胺处理的小鼠幼虫肝脏的H＆E染色nol从96 - 120 hpf。两只动物用Dietrich固定剂固定在120 hpf。 （B）中的箭头指向具有过度沉积圆形液滴的肝细胞。星号标示肿胀的肝血管。刻度棒=20μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55630/55630fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td></td><td> 10％福尔马林 </td><td> 4％PFA </td><td> Dietrich的固定剂 </td></tr><tr><td>固定条件</td><td>在室内至少24小时</td><td>在室温下3小时或4℃下的O / N </td><td>在室内至少24小时</td></tr><tr><td>样品储存柱固定</td><td>无限期在RT </td><td>在4°C达2周</td><td>最多2个月</td></tr><tr><td> Compatib与基因组DNA提取</td><td>是</td><td>是</td><td>没有</td></tr><tr><td>收缩率</td><td>是</td><td>是</td><td>最低限度</td></tr><tr><td>细胞畸变</td><td>是</td><td>是</td><td>最低限度</td></tr><tr><td>蜂窝细节丢失</td><td>是</td><td>谦虚</td><td>最低限度</td></tr><tr><td>平衡H＆E染色</td><td>弱曙红染色</td><td>弱曙红染色</td><td>是</td></tr><tr><td>与免疫组织化学的相容性</td><td>是</td><td>是</td><td>是</td></tr></tbody></table> 表1: 比较10％福尔马林，4％PFA和Dietrich的斑马鱼肝组织学固定剂。

Watch this Scientific Journal Video about Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish at JoVE.com

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

该方案描述了用2％乙醇处理24小时的斑马鱼幼虫肝脏的组织学分析。这种急性乙醇治疗导致肝血管的肝脂肪变性和肿胀。

斑马鱼急性酒精性肝损伤的组织学分析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

histological-analyses-of-acute-alcoholic-liver-injury-in-zebrafish

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

14.2K 次观看.  Cincinnati Children's Hospital Medical Center. 该方案描述了用2％乙醇处理24小时的斑马鱼幼虫肝脏的组织学分析。这种急性乙醇治疗导致肝血管的肝脂肪变性和肿胀。 

视频: Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

医学

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

癌症研究

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

行为学

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

成人斑马鱼的部分肝切除术

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

斑马鱼急性酒精性肝损伤的组织学分析

摘要

探索更多视频

此视频中的章节

肾再生的成年斑马鱼庆大霉素性损伤

在斑马鱼胚胎的机械血管损伤

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

成人斑马鱼的部分肝切除术

斑马鱼急性酒精性肝损伤的组织学分析

摘要

探索更多视频

此视频中的章节

肾再生的成年斑马鱼庆大霉素性损伤

在斑马鱼胚胎的机械血管损伤

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

在斑马鱼模型的胎儿酒精暴露评估致畸的变化

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

使用光明场显微镜定量拉瓦尔斑马鱼的肝脏大小

使用头太空气色谱对斑马鱼胚胎中的乙醇水平进行定量

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

成人斑马鱼的部分肝切除术

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生