这项工作是由一个在美国普渡大学生物科学系的启动资金支持。

第1部分：显微切割前的筹备工作<ol><li>解决方案<ol class="lalpha"><li> E3的中型4（5毫米氯化钠，0.17 mM的氯化钾， 氯化钙 0.33毫米和0.33毫米硫酸镁 ）。 </li><li>林格氏液1，5（116毫米氯化钠，氯化钾2.9毫米，1.8毫米氯化钙2，5毫米HEPES，PH7.2），过滤消毒。 </li><li> 5N氢氧化钠。 </li></ol></li><li>化学蚀刻钨针<ol class="lalpha"><li>安全小烧杯中，在培养皿中含有粘土5N氢氧化钠。 </li><li>将一侧的烧杯上的回形针，因此它与NaOH溶液接触。 </li><li>将回形针和正极了一小段的钨丝（〜2CM）的一端从直流电源的负极。 </li><li>裹的钨丝的另一端与一个小的粘土球，并为此动用的NaOH溶液。 </li><li>增加电压约为3V，DIP线和NaOH溶液中，直到一个良好的蚀刻率达到。蚀刻针质量好，通常可以在10分钟内。 </li><li>钨丝暴露的解决方案将成为逐步更薄。当粘土球脱落，另一端仍连接到正极线将有一个尖锐的针状。 </li><li>冲洗用锋利的针，林格液或E3删除盐矿床。 </li><li>将针由磁带的木制或持针器，易于处理。 </li></ol></li><li>培养板<ol class="lalpha"><li> “猎鹰”60 × 15毫米聚苯乙烯培养皿准备使用视网膜显微切割。 </li><li>对于解剖的RPE附加视网膜，彻底粉碎两个胚胎在林格氏液摆脱前30分钟清扫表面的粘附性文化板。不久之前清扫，广泛接触新鲜林格氏液洗板。 </li></ol></li><li>胚胎收集和分期<ol class="lalpha"><li>成年斑马鱼是保持为4，5。 </li><li>静态繁殖坦克独立分频器成对的亲鱼配种前的晚上。 </li><li>取出后在第二天早上打开房间的灯是分压器。允许家长交叉每隔半小时每10分钟一班。独立后，每个过路的鱼。 </li><li>从各个口岸在E3媒介收集胚胎分别保持在28 ° C。 </li><li>阶段的胚胎，在10至12小时受精后（HPF）和restage立即清扫前，在特定时间点，根据既定的准则1，6 。 </li></ol></li></ol>第2部分：夹层-去除脑部和眼睛接触1 <ul><li>本节中所述的程序共同解剖视网膜（第3A）和视网膜色素上皮附加视网膜（3B）。 </li><li>所有的精细解剖杜蒙钳尖端和定位组织的另一杜蒙钳对协助。更精细的操作，如有必要，可用于化学蚀刻钨针。 </li><li>所有的精细解剖〜5 - 8X奥林巴斯SZX16 1X的目标或相当于配备显微镜的放大下进行。 </li><li>在E3培养基中的胚胎保持在一条长凳上孵化器在28 ° C旁边的显微镜容易胚胎访问期间清扫。 </li><li>显微镜阶段由热板加热至28 ° C，以尽量减少温度的波动对基因表达的影响。 </li></ul><ol><li>在一个特定的发育阶段的胚胎转移林格猎鹰60 × 15毫米培养板中的解决方案，准备在1＃3部分描述。 </li><li>迅速切断的头部与躯干前部的一部分从身体。 </li><li>培养板引脚与协助钳这个组织后结束。 </li><li>打开背方从额头前开始的头。 </li><li>移除大脑，使眼睛的内侧暴露，并面临进一步操作上升。 </li></ol>第3A：视网膜清扫1 <ol><li>准备在第2部分所述的胚胎。 </li><li>小心地刷上面临镊子的尖端向上，直到看到一个小型开放的视网膜眼球内侧裸露的视网膜色素上皮。 </li><li>继续刷牙和脱皮的行动，直至视网膜内侧几乎全部暴露。避免刮伤和冲压暴露视网膜。 </li><li>要删除横向视网膜色素上皮视网膜，这是现在朝下，方法，刷文化板面约45 °角。 </li><li>删除是连接相对牢牢钉住的视网膜色素上皮的分离部分到C锯齿缘视网膜色素上皮ulture板夹层，然后轻轻地抬起视网膜。 </li><li>培养皿的底部滚上视网膜清理残留的RPE。 <ol class="lalpha"><li>我们注意到，这个特殊的猎鹰聚苯乙烯培养板约20分钟的视网膜色素上皮的优惠坚持，一旦胚胎被击碎。这个属性是用来清除视网膜色素上皮残余。然而，视网膜细胞的坚持程度较轻，可高放大倍率的密切合作下视察的表面。一是罢工的RPE细胞去除和视网膜完整的完整性之间的平衡。 </li><li>镜头往往坚持培养板，在轧制过程中，从视网膜分离。偶尔，它是必要的蚀刻钨针分离镜头与镜片表面上纷飞的行动。 </li></ol></li><li>这些程序，可以成功地删除从视网膜视网膜色素上皮，不影响组织的完整性。这是由良好的整体形态（图1B和B&#39;）和组织学（图1B“），尤其是箭头，保护之间的感光层和视网膜色素上皮细胞外基质是特别好（图1B。”;从整个胚胎（图1A“））与组织学比较。 </li></ol>第3B：视网膜色素上皮附加视网膜剥离3 <ol><li>准备在第2部分所述的胚胎。 </li><li>培养板，由助理钳脚的头。从外侧边轻轻地抬起眼睛，和前侧滚。 <ol class="lalpha"><li>推定脉络膜和RPE层外巩膜组织连接比较紧密，对皮肤和大部分可以去皮滚动的眼睛仔细的前侧。 </li></ol></li><li>侧方暴露的眼睛，当眼睛仍然附着在皮肤上后，钨针取出镜头。 </li><li>这些程序可以成功地保护整个视网膜RPE层。推定脉络膜和巩膜组织大体上可拆除（图1C，C“和C”）。 </li></ol>第4部分：组织的RNA工作的样品收集<ol><li>解剖样品可以收集下游RNA的特性无RNase离心管1日前在TRIZOL。 </li></ol>代表性的成果<img src="/files/ftp_upload/2028/2028fig1.jpg" alt="图1" /> 图1（一）横向（一）52 HPF前清扫了斑马鱼幼虫头部背面观。 （A“），相应的幼虫头部的组织切片（A）及（一）（二）横向和（B&#39;），在54 HPF解剖视网膜背视图。视网膜表面是完整的两个横向背的意见（二）相应的病理解剖视网膜节（b）及（B“）。视网膜和视网膜层压结构完整。特别是细胞外基质之间的感光层和RPE（一“，箭头），保存完好，在解剖视网膜（B”箭头）。 （三）横向（C“）52 HPF的视网膜色素上皮附着在视网膜的一个解剖内侧。 RPE层是完整的和连续，这也是由病理解剖组织的第（三）表示。“C”的白色区域是视神经（箭头），组织学，组织标本在4％，收集和固定多聚甲醛塑料包埋和切片对这些样品进行了描述。比例尺= 50微米。

<ol>
	<li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retina.">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li><li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+microarray+analysis+of+zebrafish+retinal+development.">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li><li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo.">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish : a practical approach. , (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+book+:+a+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio).</a> , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li></ol>

普渡大学动物护理和使用委员会规定的准则和法规的规定进行了动物实验。

斑马鱼眼组织显微切割可以有效地获得完整的视网膜和视网膜色素上皮附加视网膜。这大大助攻表达的研究，涉及到一个特定的眼组织（即视网膜色素上皮）。事实上，我们已经成功地利用这些程序获得整个视网膜和视网膜色素上皮 3 RNA的表达谱。这些配置文件的实用工具，是我们最近的途径和基因家族在视网膜分化突变2扰动识别，大力支持。在视网膜的解剖程序的最关键步骤?...

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cordless pestle motor</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-370</td>
<td>&nbsp;</td>
<tr>
<td>DC power supply</td>
<td>&nbsp;</td>
<td>Lascar</td>
<td>PSU130</td>
<td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td>
</tr>
<tr>
<td>Disposable pestle &amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-366</td>
<td>These are used for tissue collection in TRIzol for expression analysis.</td>
</tr>
<tr>
<td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500341</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Falcon polystyrene culture plates, 60 X 15 mm</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351007</td>
<td>These plates are used as dissection plates.</td>
</tr>
<tr>
<td>Olympus SZX16 Stereomicroscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX16</td>
<td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td>
</tr>
<tr>
<td>Sharpening stone</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>29008-01</td>
<td>Use this to sharpen the tip of the forceps if necessary</td>
</tr>
<tr>
<td>Thermo plate</td>
<td>&nbsp;</td>
<td>Tokai Hit</td>
<td>MATS-U55SZX2B</td>
<td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&deg;C during dissection in previous studies1-3 with good success.</td>
</tr>
<tr>
<td>Trizol, 100 mL</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>tungsten wire, 0.015 inch diameter</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TGW1510</td>
<td>&nbsp;</td>
<tr>
<td>Wooden Applicator</td>
<td>&nbsp;</td>
<td>Puritan</td>
<td>807</td>
<td>This is used for holding the chemically-etched tungsten needle.</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of zebrafish embryonic eye tissues

斑马鱼眼睛发育的模式，因为其快速流行的动物进行研究<em&gt;前宫内</em&gt;发展和良好的繁殖力。受精后3天（DPF），幼虫会显示第一视觉反应。许多基因已被确定为控制适当的眼球发育，但我们正在从一个潜在的遗传结构的完整的理解远。全基因组基因表达分析是一个有用的工具，以澄清对眼睛发育的基因调控网络。然而，在斑马鱼的胚胎眼的小尺寸使得它具有挑战性的，以取得完整和纯粹的眼部组织表达分析。例如，2和第3天之间的眼睛前后的长度仅约为200-300微米，而镜头的直径为100微米少。此外，基本视网膜视网膜色素上皮（RPE）仅仅是一个单层上皮。虽然可以从整个胚胎中获得的基因的表达谱，他们不准确地代表这些组织中的表达。因此，纯粹的组织必须得到一个成功的眼睛发育的基因表达图谱。为了解决这个问题，我们已开发出一种方法来microdissect附1-3 DPF与视网膜色素上皮完整的视网膜和视网膜，其中包括眼的形态发生的主要阶段。所有的程序可以做到的标准立体显微镜下用细镊子和一般实验室用品。视网膜剥离，单层RPE细胞被删除，去皮刷牙行动和培养板剥离面的视网膜色素上皮残余的优惠坚持。对于视网膜色素上皮附加视网膜剥离，视网膜色素上皮坚持夹层板被删除之前，剥离，使RPE细胞可以与视网膜完全保留。一个小心解除这个组织的行动能够有效地分离推定脉络膜和巩膜。镜头可以在这两种情况下，由化学蚀刻钨针删除。总之，我们的方法可以获取完整的眼部组织，并已成功地利用研究斑马鱼视网膜组织特异性表达谱<sup&gt; 1，2</sup&gt;和视网膜色素上皮细胞<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

Microdissection of Zebrafish Embryonic Eye Tissues

本文介绍的方法microdissect与视网膜色素上皮细胞附着，从一到三天受精胚胎斑马鱼视网膜。

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 ...

microdissection-of-zebrafish-embryonic-eye-tissues

Research

JoVE Journal

Biology

16.0K 次观看.  Purdue University. 本文介绍的方法microdissect与视网膜色素上皮细胞附着，从一到三天受精胚胎斑马鱼视网膜。

视频: Microdissection of Zebrafish Embryonic Eye Tissues

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

显微切割的黑寡妇蜘蛛丝绸生产腺体

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...