This work was supported by NIH grant F32DK091998 to CNK; NIH grant RO1DK041071 and Harvard Stem Cell Institute grant D001229 to IAD. The authors thank Neil Hukriede for the lhx1ain situ probe.

注：伦理学声明：所有实验均按照用于研究使用动物马萨诸塞州总医院的指导方针进行。 1.事前准备<ol><li>确定有多少成年斑马鱼6-12个月大的伤害。计划伤害10-20％以上的鱼比是必要的。为了尽量减少可变性，使用年龄匹配的同胞的鱼饲养在同一个罐，使它们大致相同的尺寸，并具有更少的遗传变异。母鱼更容易注入相比公鱼，由于腹部的更大的容量。但是，男性和女性给予了类似的结果。 </li><li>当做实验首次，绘制剂量曲线，以确定适当庆大霉素剂量为鱼的特定菌株。在实验中使用之前测试每一批新的庆大霉素庆大霉素以来的纯度一批不同于一批。适当的剂量应通过评估expressi确定上的损伤标记物如lhx1a。 （参见图1A - D）。 </li><li>准备庆大霉素解决方案。庆大霉素原液可被存储在-20℃，解冻后使用。例如，对于一个80毫克/公斤的剂量（80毫克庆大霉素/ kg体重的）在一个鱼体重0.5克，制备庆大霉素2毫克/毫升在磷酸盐缓冲盐水作为一种方便的工作溶液。这提供了一个20微升庆大霉素为腹腔注射成鱼。 注：在一般情况下，80-120毫克/千克取得良好的效果，但剂量低至40毫克/公斤，可适当。庆大霉素也可在溶液中购买，以尽量减少有害暴露于实验室人员。 注：庆大霉素高剂量可能是有毒的。戴上手套，重粉面膜的时候。 </li><li>制备100毫升0.016％三卡因水用于麻醉鱼。制备一个25倍的库存三卡因（4克/ 1L）溶于无菌milliQ水中的调节至pH 7，并储存在4℃直至使用。淡化4米升在100毫升鱼水的0.016％工作浓度。 注：三卡因是一种麻醉剂和皮肤有刺激性，搬运时戴上手套。 </li><li>做鱼舀出塑料移液管的切割灯泡成勺形和切割2槽底部排水。负载1毫升注射器（带10微升等级）与庆大霉素准备解决方案，并附加一个30½摹针。去除气泡。拧上的注射器针头，以确保在针的斜角尖背向和针筒标记是朝前和可读性。对于控制注射，准备一个注射器和针头用无菌PBS。 </li><li>准备一个规模用干净的表面或船的重量，以及纸巾晒鱼和鱼保持注射。 </li><li>准备个人的小半升有盖的容器举行的鱼一夜之间观察注射后。小透明塑料笼交配是用于此目的的。这些容器不应白色 - 理想，他们应该清楚，这样的鱼棚白色上皮管型在黑色的台面进行观察。填写每个容器有足够的鱼水的鱼游泳舒适。 </li></ol>庆大霉素2.腹腔注射<ol><li>在麻醉鱼水0.016％三卡因溶液的鱼。等待鳃通风量，以减缓和鱼不再响应触摸。 </li><li>舀起使用鱼舀鱼，从头部向尾部，以避免伤及鳃鳍或移动。放在纸巾鱼通过在其一侧轻轻转动捞出来吸收过量的水，从勺甩掉多余的水份。 </li><li>再舀上来的鱼，并把它在权衡船在归零规模和权衡鱼。一轮以最接近0.25克，并计算庆大霉素注射适量。 注：0.5g的鱼用20微升注射80毫克/公斤的剂量。 </li><li>再舀上来的鱼，并把它放在一个干燥的折叠纸巾。如果用右手注入，按住纸巾在左手，并把鱼的头指向左边，用肚皮方便。 </li><li>握住针注射器以45°角，腹部皮肤，先于泄殖腔。按刚下皮肤进针，然后下降的角度和向前滑动针在皮肤下，避免内脏。压低柱塞适量，暂停，以确保没有液体出来时分针，然后拔出针头。如果拿着鱼稳定是一个问题，对躯干肘部支撑，以稳定他们。 注：当处理庆大霉素溶液戴上手套。 </li><li>每条鱼落入一个单独的容器中。观察鱼，并保证从麻醉中复苏。保持鱼在28.5℃下过夜。 注：如果鱼没有立即恢复，使用反胶FER吸管灌溉水在其鳃来恢复它。 </li><li>当用相同剂量庆大霉素注入多个鱼使用相同的注射器和针头。处理用过的注射器和针头插入相应的生物危害锐器容器。 </li></ol>损伤3.注射后观察<ol><li>第二天（1天伤后），放置在其容器注入的鱼放在一个黑暗的表面。死亡的上皮组织中受伤的鱼排出的白色塑像应该是可见的。 （参见图1E - H）。如果没有管型，无论是鱼由庆大霉素注射没有受伤（或者剂量太低，或者一些在注射过程中泄漏的庆大霉素）或鱼严重受伤导致输尿管和泄殖腔的完全阻断与脱落的组织。 注：如果鱼是无法清除其体内的塑像它通常会在2-3天内死亡，因此不能用于再测定。如果没有强制转换是可见的，通过浸渍至少10分钟鳃运动停止后安乐死在三卡因水中的鱼。 </li><li>如果白石膏组织可见，抛开鱼并继续检查其他鱼类。庆大霉素的适当的剂量将导致鱼是可用的80-90％。池鱼受伤，然后将它们如果需要分成不同的治疗组。 </li></ol> 4.护理恢复的鱼<ol><li>保持鱼在清水和拥挤的环境。脏水和拥挤会导致感染和意外死亡。如果可能，尝试每天保持不超过6尾/500毫升鱼水和换水。 注意：这是一个最小的体积为节约空间或如果研究者希望执行使用昂贵的药物治疗实验。 </li><li>不要喂的鱼，直到后3天的伤害。恢复鱼不会吃第一，并在坦克任何食品会发生分解，并促进细菌克owth。第3天开始伤后，每天喂一次少量。 </li></ol>肾脏损伤5.分析<ol><li>对于为 lhx1a表达原位杂交，肾脏可以从鱼都收获希望的时间点。冰在三卡因水中鱼实施安乐死，至少10分钟，直至鳃运动停止，然后取出头用刀片并打开体腔，并用钳子取出内脏。 </li><li>留在原地（贴在背体壁色素器官），肾和PBS固定在4％多聚甲醛鱼过夜。解剖出肾15并继续原位杂交标准。 <ol><li>简要地说，洗净肾脏在PBST（磷酸盐缓冲盐水0.5％吐温20），进行1小时，在室温下透化用蛋白酶K在PBST（10微克/毫升）。用4％多聚甲醛后固定，并用PBST再洗涤。 </li><li>然后预杂交肾脏过夜68;℃下在杂交缓冲液（50％甲酰胺，5×SSC，50微克/毫升肝素，500微克/毫升的tRNA，0.1％吐温20，pH 6.0）中。 </li><li>孵育在杂交缓冲液中地高辛标记探针的样品中，然后再洗涤5-10分钟每次用100％，75％，50％，25％，杂交缓冲液在68℃，然后转移到室温，并清洗两次，每次30分钟，用2×SSC，0.1％吐温20，和清洗两次，每次30分钟，用0.​​2×SSC，0.1％吐温20。 </li><li>平衡样品3×5分钟，在生物圈（0.1M马来酸，0.15M氯化钠，pH值7.5）和封闭过夜，在4℃在生物圈用10％山羊血清。然后孵育样本过夜抗地高辛-AP的Fab 1：5000人与生物圈。 </li><li>洗样品5倍1小时的人与生物圈计划，并平衡3倍于NTMT 15分钟（0.1米的Tris pH9.5，0.05M的氯化镁 ，0.01 M氯化钠，50微升吐温20）。 </li><li>然后对待NBT / BCIP肾脏检测信号。固定用4％多聚甲醛的肾脏并孵育在二甲基甲酰胺字符型数据已经超额NBT / BCIP，在去离子水中漂白过夜。洗肾脏，然后拍摄在PBST中与下一个盖玻片50％甘油。 </li></ol></li></ol>

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The authors have no competing financial interests to disclose.

斑马鱼是理想的学习成人器官再生包括成人中肾肾脏6。最近的研究已经利用分子标记技术和转基因新记者行更好地表征在肾再生发生的步骤，哪些细胞可能是负责7,12。尿管型的观察已使用了一个多世纪来诊断肾脏疾病在人类16。在这里，我们使用管型的存在，作为庆大霉素注射后一种简单，非侵入性的和早期指标的急性肾损伤的程度。我们观察到的鱼，不产生强制类型转...

肾脏是流体平衡，血压调节和过滤废物从体内必需的。虽然哺乳动物可以利用受伤分化的上皮细胞后1-4修复肾单位存在，他们似乎缺乏保留的干细胞5的游泳池，并不能形成新的肾单位从头 。在对比哺乳动物，成鱼能够形成整个成年生活新肾支持鱼的生长，并且响应于损伤6,7。斑马鱼， 斑马鱼 ，是一种宝贵的模式生物器官再生8-10的研究，并有可能提供强大的洞察到应用程序中来改造人类的肾脏的修复。 的Tg（Lhx1a：EGFP）转基因11已经显示然而标记肾祖细胞的池，在成年斑马鱼肾12，控制lhx1a的响应+细胞损伤ř机制emain不清楚。 氨基糖甙类的庆大霉素是一种广泛使用抗生素与人类已知的13和肾耳毒性作用。腹腔注射庆大霉素是一家成立于鱼6诱发急性肾损伤的方法。这种损伤的鱼模仿的肾小管上皮细胞，而发生在人类过量服用庆大霉素后14肾小球疤痕的损失。斑马鱼中的庆大霉素注射诱导损伤是引起强烈的，同步再生反应，产生了许多新的肾单位，同时继续通过形成，增殖和分化阶段的便捷方式。 该协议通过利用无创可视化的筛选过程中尽量减少离群点细节我们的方法可再生的损伤的成年斑马鱼肾脏。我们注意到，与庆大霉素损伤导致的肾上皮组织和格式死亡的事实优势离子肾小管演员，然后积聚成群众中肾管和泄殖腔。这些是由鱼传递，并且可以在水目视观察。这使我们能够对鱼类具有较强的非致命伤害，然后可以为汇集进一步的实验筛选。最小化的未受伤的鱼或鱼，才到达实验的终点导致更均匀和高效的数据收集和分析该模具的数量。此外，没有任何特殊的设备或试剂要求，使得这种方法具有成本效益，并适用于在学术或教学环境中使用。使用我们的方法，在这里，我们表明肾再生庆大霉素剂量的增加效果以及增加温度的作用。

<table border="0" cellpadding="0" cellspacing="0" width="726">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:101px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:291px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin sodium sulfate</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">G1264</td>
			<td>TOXIC, purity varies from batch to batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic transfer pipets</td>
			<td style="width:101px;">Fisher</td>
			<td style="width:119px;">13-711-7M</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">1 ml Norm-Ject syringes</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">72520</td>
			<td>green plastic syringes, ordinary 1ml syringes are OK, but harder to read accurately</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">30G1/2 needles</td>
			<td style="width:101px;">Becton Dickinson</td>
			<td align="right" style="width:119px;">305106</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ethyl 3-aminobenzoaate methanesulfonate salt (tricaine)</td>
			<td style="width:101px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>IRRITANT</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">16% paraformaldehyde</td>
			<td style="width:101px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td>Make 4% in 1xPBS for working solution</td>
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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.

庆大霉素损伤可目视由肾上皮管型的观察中的水（ 图1）的确认。庆大霉素不同剂量被用来伤害野生成年斑马鱼TuAB，导致在增加再生肾脏lhx1a +细胞聚集（ 图1A - D）的数字。白石膏可以很容易地看出，在水下1伤天之后（ 图1E - H）。庆大霉素低剂量导致不具有强制类型转换大多数鱼，而高剂量导致了鱼类有管型（ 图1I）</s...

Watch this Scientific Journal Video about Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury at JoVE.com

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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 ...

kidney-regeneration-in-adult-zebrafish-by-gentamicin-induced-injury

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  Massachusetts General Hospital. 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. 

视频: Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

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 ...

科研

发育生物学

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 ...

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 ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

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 morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

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 ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

纳米粒子介导的 siRNA 基因沉默在斑马鱼心脏中的表达

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

成年斑马鱼端脑中疟原虫DNA在活体传递的电穿孔方法

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...