该协议已经制定了与麦金尼斯实验室，金A.锤和约瑟夫·皮尔逊的早期成员合作。我们感谢继续努力的布鲁明顿果蝇库存中心为他们的工作，组织和分发有价值的股票。这项工作是由美国国立卫生研究院（R01 GM077197和K12 GM68524）和赫伯特·斯特恩的家庭提供资金支持。 MTJ目前由授权号5G12RR003060-26来自国家研究资源中心与授权号码8G12MD7603-27从国家学院少数民族健康与健康差距的支持。内容完全是作者的责任，并不一定代表研究所少数民族健康与健康差距和美国国立卫生研究院的官方意见。

果蝇胚胎伤口协议可以概括为五个步骤：（1）胚胎收集，（2）胚的制备（3）胚胎伤人，（4）胚胎显微注射，和（5）胚胎固定。 1。胚胎收集<ol><li>收集成蝇，eclosing后2-3天，加上50名女性和25位男性到胚胎收集笼。 </li><li>每天早晨将一个新鲜的苹果汁琼脂加酵母片3天。 注：为了获得最佳的胚胎收集，循环在25℃培养箱中培养12小时昼夜时间表苍蝇（05:00灯“ON”和17:00灯“关”）;雌性果蝇作出回应的变化，光周期和将从灯光开关转到“ON”，以灯为“OFF”10,11中存入大批量的鸡蛋。 </li><li>在第3天下午，放置一个新鲜的苹果汁加酵母琼脂平板上笼，收集胚胎2小时。将新的酵母片上笼并保存2小时收集板。 </li><li>存储该收集板为额外的14小时，在25℃下老化所述胚胎。 注：伤口愈合可以在任何阶段果蝇发育过程中检测，在本文中所描述的伤口诱导的转录记者们的阶段一十二月15 日至16日之间的最佳活动。通过后期17，胚胎是太老了，容易穿透角质层成熟而有效地激活伤口记者或检测伤口诱导的转录。要允许发生的集合时间和伤人时间在繁忙的实验室小时，我们按照胚胎收集的2小时（16:00-18:00）的时间表，在18:00交换新盘收集板，年龄收集板在25℃下14小时（过夜），和（第二天）伤口的胚胎在08:00。 </li></ol> 2。胚胎的制备注：此协议包括使用许多工具和对象是尖锐或作出克姑娘。处理所有的锐器，并小心玻璃物体，以免造成人身伤害。 <ol><li>轻轻地加入2-3毫升水盘和旋转从一个画笔收集盘取出胚胎。从板块转移水和胚胎尼龙网和收集管。 </li><li>加4-5毫升的漂白剂在塑料陪替氏培养皿盘的盖和浸泡在含有漂白剂的胚胎，持续2分钟收集管的网状覆盖端;除去胚的外蛋壳。手动旋转收集管几次，以防止胚胎结块的漂白剂。胚胎冲洗用水10倍和干含在纸巾上的dechorionated胚胎网格覆盖的收集管。 </li><li>用解剖针转〜50胚从尼龙网到琼脂平板。我们增加了绿色食用色素的琼脂平板加对比度和援助胚胎对齐。在解剖显微镜下，使两排平行〜25的胚胎，对准安莉芳口上滑动，以便它们将是成直角的注射装置中的穿刺针。留下一点空间胚胎进行气体交换的目的之间（约1胚胎长度）。在两个平行的行应该是大约5毫米。 </li><li>将幻灯片与双节棍磁带上的排列胚胎的顶部，小心地按幻灯片从绿盘转移胚胎到幻灯片中。然后在空气中干燥的胚胎〜25分钟，以减少对胚胎的内部压力，并防止突发伤人时。 </li><li>将2-3滴卤烃油混合在胚胎，以防止进一步脱水。 </li></ol> 3。胚胎伤人<ol><li>放置在注射显微镜阶段胚胎的幻灯片。发现胚胎在视场和练习移动倒置显微镜的阶段。 注：在用针工作，专注于沿背腹轴的胚胎中面。允许的有效伤人对齐的胚胎，开始伤人的行靠近针头装置。移动载物台，使对准胚胎的行的顶部进入视场。 </li><li>小心地放置和固定拉针在针托。使用显微操纵器移动针朝上下滑动。对准胚针，在同一焦平面上。一旦针是关注的焦点与胚胎，不要移动与显微操作针。 </li><li>移动台通过，并通过穿刺胚胎，然后转移到该行中的下一个胚胎。完成后伤人的第一行，用显微操作来从舞台移动幻灯片之前完全移动针和远离;手动旋转滑盖180°和伤口的第二行。 </li><li>根据存储卤烃油的胚胎在室温下进行胚胎固定，如果做原位杂交或抗体染色（见程序步骤5）。另外，存储胚胎4-6小时在卤烃油进行伤口转录荧光报告的可视化（代表结果）。 注意：对于荧光报告的可视化，我们麻醉用50％的1 -苯氧基-2 -丙醇13的胚胎。将胚胎从卤代烃油中取出并放在一个新的幻灯片。我们把玻璃珠周围的胚胎，加几滴麻醉，并放置在胚胎盖玻片。 </li></ol> 4。胚胎显微注射注：我们在协议中使用的注射装置不是自动的在试验过程中提供特定的音量。我们的染料添加到溶液中以可视化的显微注射，并尝试正常化递送的体积。如果太多的溶液注入胚胎，卵黄膜会破裂。 <ol><li>负载针从背面用1微升的化学溶液加染料（ 例如 0.6 MH 2 O 2）。允许毛细管动作来绘制化学溶液注射针的尖端。 </li><li>为了打破一针，放置盖玻片在一个角度上一张幻灯片，然后涂上卤烃油混合的边缘。把针头装成焦点与角度的盖玻片的边缘，轻轻移动盖玻片边缘穿过针的尖端，直到破裂。施加压力的显微注射设备，并检查药液加染料流出针。 </li><li>将包含在显微镜舞台对齐胚胎的幻灯片。继续集中在同一平面内的胚胎和针。同时穿刺和化学微导液加染到每个胚胎。 注：针头堵塞经常发生，闯出了一条新针，以避免不完整的显微注射。钝针尖不作为伤口清洁是一个倾斜的针尖。调整盖玻片到非90°角破损时是最佳的，以产生一个带角度的针尖。 </li></ol> 5。甲醛网络xation 注：在此果蝇固定使用的化学品是有害的。处理化学品时使用个人防护装备（ 如手套，实验室外套，护目镜）。按照处置化学品监管个别机构的指导方针。 <ol><li>伤人后，等待15，30，60，分钟或更长时间的伤口诱导的基因发生了转录激活。若要从幻灯片注射胚胎取出，小心地从滑梯沥干卤烃油混合，靠在机架的幻灯片，用Kimwipe擦拭吸干结束。 </li><li>使用玻璃移液管将清洗的庚烷在滑动和漂洗液洗入玻璃培养皿。庚烷将解散磁带和释放胚胎。继续漂洗，直到所有的胚从滑动取出。进入到下一张幻灯片，直到所有的胚胎从幻灯片中删除。 </li><li>转让从玻璃盘庚烷加胚液放入20 ml SCIntillation小瓶。摇动小瓶2-3分钟，取出庚烷，加入5毫升新鲜庚烷。 </li><li>加入5毫升新鲜的，固定的解决方案（见名单试剂配方）。盖上瓶盖，并连接到一个轨道摇床。摇含有胚胎在220-230转25分钟的小瓶。 </li><li>摇匀后，让气泡在界面上弹出。完全用吸管除去底部含水相，避免拉起胚胎。底层是由固定的解决方案，并应妥善处置。 </li><li>加甲醇5ml，盖上瓶盖，并用手用力摇动20-30秒，漩涡，并将其放置在板凳上。受伤的胚胎会留在间期，也不会沉到水底。 注：固定后的未受伤的胚胎的卵黄膜在脱水步骤用甲醇将正常突发。然而，在一个受伤的胚胎的卵黄膜已经被打破，脱水步骤不会删除卵黄MEMBRANE。手devitellinzation必须完成固定的协议。 </li><li>小心地取下顶部庚烷层，然后加入5毫升新鲜甲醇。摇瓶和胚胎应该下沉。 </li><li>除去甲醇加胚胎溶液，用玻璃移液管和收集在玻璃培养皿的尼龙筛收集管。 </li><li>除去甲醇和冲洗用1×PBS溶液中的胚胎。 </li><li>从尼龙网胚胎转移到苹果汁板。传播胚胎沿所述​​板的表面上。 </li><li>胚胎转移到一个双面胶带搏命。覆盖用1×PBS中的胚胎。 注：胚胎往往因为卵黄膜的结块。用苹果汁的地方允许成群胚胎的快速分离。胚胎必须从板中移除，并转移到一个两面胶带载玻片因为胚胎沉入琼脂平板上时，力被施加到从取出胚胎卵黄膜。 </li><li>小心地将胚胎用解剖针“啪”的卵黄膜。胚胎应该沉沦在PBS中。 </li><li>使用玻璃移液管的1×PBS中加胚胎转移到1.5ml管中。冲洗用1×PBS和存储的胚胎在4℃下继续进行脱水用乙醇胚胎：PBS系列，并继续与原位杂交或免疫定位协议14。 </li></ol>

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(63), e3347 (2012).</li><li>Repiso, A., Berganti&#241;os, C., Corominas, M., Serras, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+repair+and+regeneration+in+Drosophila+imaginal+discs.">Tissue repair and regeneration in Drosophila imaginal discs.</a> Dev. Growth Differ. 53 (2), 177-185 (2011).</li><li>Worley, M. I., Setiawan, L., Hariharan, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+and+transdetermination+in+Drosophila+imaginal+discs.">Regeneration and transdetermination in Drosophila imaginal discs.</a> Annu. Rev. Genet. 46, 289-310 (2012).</li></ol>

没有利益冲突的声明。

到外部线索立即转录调控响应允许细胞坐标的局部响应于细胞外刺激，其可以包括应力，损伤或感染。不当控制局部反应可能对相邻小区的破坏性影响，以及资源的浪费，以修复损伤。 果蝇胚胎提供了极好的系统，以在一个廉价的和易于维护的模型生物体进行基本的伤口愈合试验。在其他模式生物的研究和果蝇之间的合作潜力提供了一个独特的机会去探索许多生物学问题。 伤口记者的附加技术是突变体背景的基因组合，提供了用于测试的新的基因，以表皮伤口反应途径的活化的贡献的有效方法。我们有表皮伤口诱导的转录记者AVailable在2号，3号染色体5两者。在这项研究中，我们使用无人机和表达16 GAL4系统。对于致死突变等位基因的研究，我们决定使用荧光平衡器染色体， 如Kruppel-GFP 17的遗传背景。我们已经分析了几种遗传背景（ 表1）伤口记者本土化。 该技术的一个可能的修改是结合显微注射和伤人。显微注射可以用来引入一个化学溶液注入胚胎。几种化学成分进 ​​行了测试，发现它们调节表皮伤口记者9的活性。同时伤人和显微注射的这个方法可以是有用的，以增加细胞在果蝇胚胎进行了回复伤信号，可直接用于监测基因表达的变化伤人18后的数。显微注射技术的应用前景将是测试新的化学品的表皮创伤反应的调节。 果蝇作为遗传学研究的模式系统提供了多种简单的协议，有效地解决复杂的问题。对果蝇技术的可行性最近的报告强调了使用血细胞迁移19和寄生蜂感染20为手段，以进一步拓宽果蝇研究界的影响。除了 ​​创伤修复的研究， 果蝇提供了一个经典模型进行再生与成虫盘系统21,22。在成虫盘再生的研究和穿刺/注射伤人合并的进展提供了一个很好的系统来发现组织修复以及保守的成分。

用显微注射技术果蝇胚胎的操控是一个行之有效的分析1。表皮伤口响应报道方法的意义在于对协议 ​​进行穿刺/注射结合荧光报告和可视化的体内响应于表皮损伤的果蝇胚胎。该方法的目的是使一组科学家更广泛的使用果蝇作为一种工具，以调查，调节表皮穿刺伤的转录反应的进程。单层表皮果蝇提供了一个简单系统中的穿刺伤2后，研究表皮伤口响应。 果蝇胚胎是一个强大的模型系统进行遗传解剖的伤口修复的阶段，包括伤口反应，炎症和表皮细胞再生3。这部分是因为许多或大多数合子突变体存活到embryogenes的端是发展表皮屏障，即使发育模式去深刻出差错。以前表征一个高度保守的转录因子粒状头（GRH），确定该GRH-靶基因的多巴脱羧酶（DDC）和酪氨酸羟化酶（PLE）的转录周围损伤4的位点被激活。 果蝇作为模式生物用于伤口响应提供调查的补充，以推进研究在哺乳动物伤口愈合5。随后的研究发现有其他果蝇伤口诱导的基因，并制定了“工具箱”的许多荧光伤口记者来监视体内表皮伤口的反应来清洁伤人6。对果蝇伤口愈合的调控最近的报道都集中在伤口闭合型，允许探索多种途径调节细胞迁移7,8。用的分析荧光伤口记者，我们的研究已经确定了一套所需的表皮伤口诱导基因9本地化表达基因的小说。一项所述的伤口响应报道方法的优点是，该结果提供了更深入的信号是如何从损伤部位转导到相邻小区的机制。然而，在伤口响应报道方法的缺点之一是，结果不直接链接到在伤口愈合或修复的表型。在遗传和化学屏幕更广泛地利用清洁伤口穿刺协议将允许转录反应的新的监管机构，以表皮伤人被识别并进一步伤口愈合发现翻译成伤治疗哺乳动物模型。

<table border="0" cellpadding="0" cellspacing="0" width="576">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">yeast</td>
			<td style="width:124px;">Sigma Aldrich</td>
			<td style="width:133px;">51475</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">apple juice</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">agar</td>
			<td>Fisher Scientific</td>
			<td>50-824-297</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bleach</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 700 W</td>
			<td>Sigma Aldrich</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 27 W</td>
			<td>Sigma Aldrich</td>
			<td>H8773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1-phenoxy-2-propanol</td>
			<td>Sigma Aldrich</td>
			<td>484423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydrogen peroxide</td>
			<td>Fisher Scientific</td>
			<td>H324</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methyl-&#946;-cyclodextrin</td>
			<td>Sigma Aldrich</td>
			<td>C4555</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">toluidine blue</td>
			<td>Sigma Aldrich</td>
			<td>89640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formaldehyde, 16%</td>
			<td>Polysciences, Inc</td>
			<td>18814-20</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">heptane</td>
			<td>Fisher Scientific</td>
			<td>H360-1</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methanol</td>
			<td>Fisher Scientific</td>
			<td>A412-1</td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>Fisher Scientific</td>
			<td>50-899-90013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EGTA</td>
			<td>Fisher Scientific</td>
			<td>50-255-956</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RNase free H2O</td>
			<td>Fisher Scientific</td>
			<td>BP2819-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:121px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila incubator</td>
			<td>Genessee Scientific</td>
			<td>59-197</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fly cage</td>
			<td>Genessee Scientific</td>
			<td>59-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">embryo collection tube</td>
			<td>Genessee Scientific</td>
			<td>46-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">plastic Petri dish</td>
			<td>Fisher Scientific</td>
			<td>50-202-037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">double-stick tape</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">paintbrush</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissection needle</td>
			<td>Fisher Scientific</td>
			<td>08-965A</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass cover slip</td>
			<td>Thermo Scientific</td>
			<td>3306</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microscope slide</td>
			<td>Thermo Scientific</td>
			<td>4445</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting microscope</td>
			<td>Carl Zeiss</td>
			<td>Stemi-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">capillary needle</td>
			<td>FHC</td>
			<td>30-30-1</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">needle puller</td>
			<td>Narishige</td>
			<td>PC10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microinjection needle holder</td>
			<td>Narishige</td>
			<td>MINJ4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">micromanipulator</td>
			<td>Narishige</td>
			<td>MN151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">inverted microscope</td>
			<td>Carl Zeiss</td>
			<td>Primo-Vert</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747A</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>22-063-172</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">transfer bulb</td>
			<td>Fisher Scientific</td>
			<td>03-448-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kimwipe</td>
			<td>Fisher Scientific</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">scintillation vial</td>
			<td>Fisher Scientific</td>
			<td>03-337-7</td>
			<td>sharp object</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">orbital shaker</td>
			<td>Fisher Scientific</td>
			<td style="width:133px;">14-259-260</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser scanning confocal</td>
			<td>Leica</td>
			<td>SP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fixation solution</td>
			<td>(5 ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">16% formaldehyde</td>
			<td>2.5 ml</td>
			<td></td>
			<td>toxic chemical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x PBS</td>
			<td>0.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EGTA</td>
			<td>0.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RNase free H2O</td>
			<td>1.5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Halocarbon oil mix</td>
			<td>(10 ml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 700 W</td>
			<td>5 ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">halocarbon oil, 27 W</td>
			<td>5 ml</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

microinjection wound assay and in vivo localization of epidermal wound response reporters in drosophila embryos.

果蝇胚胎发育，供应既保护了内部的细胞从一个严峻的外部环境以及维持细胞稳态强大的表皮层。穿刺伤用玻璃针提供了一种直接的方法来触发快速表皮伤口响应，激活伤口转录记者，这可以通过一个局部记者信号在活胚胎或幼虫被可视化。穿刺或激光的伤害还规定，促进血细胞的招募到伤口部位的信号。出人意料的是，重度（通过，并通过）穿刺损伤后期胚胎只有很少干扰正常胚胎发育，如大于90％，例如受伤胚胎存活到成年时的胚胎中，最大限度地减少直接泄漏血淋巴中的从穿刺部位的油介质被注入。卷绕过程确实需要显微操作果蝇胚胎，其中胚胎在银手动对准AR板和对齐胚胎显微镜载玻片转移。 果蝇表皮伤口响应分析提供了一种快速的系统进行测试的各种生物学功能，促进伤口愈合，以及一种方法来筛选潜在的化学化合物，其促进伤口愈合的基因的需求。生命周期短，易培养例行使果蝇的一个功能强大的模式生物。 果蝇清洁伤口愈合出现协调表皮再生反应，与先天免疫反应，在方式，则仍在调查中，它提供了一个很好的系统来找到保守的监管常见的果蝇和哺乳动物表皮伤人机制。

取得在该方法中所示的结果，绿色荧光蛋白（GFP）的开放阅读框融合至从多巴脱羧酶（DDC）和红色荧光蛋白融合到从酪氨酸羟化酶伤增强子序列（启动子/伤口诱导的增强子序列PLE）4,6。在一个活果蝇胚胎中，荧光伤口报告蛋白的成熟过程是最优的4-6小时后伤人，这使得时间的足够的蛋白质的积累来可视化，以及作为时间的荧光蛋白氧化的荧光状态15。观察记者本地化，化合物荧光显微镜是足够的。观察记者本地化的更高的放大倍率，在共聚焦显微镜下是最优的，生成的多个光纤段的最大凸起（ 图1）。共聚焦在果蝇胚胎的体内成像是由RA并发胚胎的PID起伏。 。可以用20X物镜来获得一个完整的视图胚胎，可以用40倍物镜获得伤口部位的特写视图。一顶视图，表皮的伤口记者本土化可以成像与10连续1μm光学部分。一种横向视图表皮伤口记者本土化可以成像与40连续1微米的光学部分。 使用标准遗传交叉方法中，有可能伤记者结合的基因突变。其中一个例子本文提出，是因为（以P元素插入产生无效等位基因）功能丧失和增益的功能- （无处不在的表达在所有细胞中产生的过表达）的突变体Flotillin-2（FLO-2）， FLO-2演示型Flo-2基因产物是必要的和足够的抑制DDC-GFP的定位卷绕记者9（ 图2A和2B）。使用英里croinjection协议，它可以测试引入的化学溶液superactivates还是抑制伤口记者的本地化。化学伤员胚胎同时受伤和注射1:4的比例为1％甲苯胺蓝染料和增溶​​的化合物。甲苯胺蓝染料可用于增溶的化合物被注入到体腔内的视觉确认。控制胚胎受伤了断针含1:4的比例为1％甲苯胺蓝染色和溶质无化学。本文所提出的两个例子是过氧化氢 ​​（H 2 O 2）和甲基-β-环糊精（MβCD），因为这两种化学溶液是足够用来全局启动DDC-GFP的定位卷绕记者9（ 图2C和2D） 。过氧化氢 ​​（H 2 O 2）溶液稀释于H 2 O至0.6米甲基-β-环糊精（MβCD）溶解于1米M氢氧化钠至3毫米。使用胚胎固定方案，它能够检测的卷绕响应基因的转录活化中的局部领域，围绕伤口部位。一个例子在本文提出的是在 RNA探针原位杂交检测DDC和辉成绩单4,6,9（ 图3A和3B）。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50750/50750fig1.jpg" width="600px" /> 图1。 DDC-GFP和PLE-DsRed的伤口记者本土化。明场和荧光图像中受伤的野生型胚胎（A） -顶视图DDC-GFP伤记者（B） -顶视图PLE-红色荧光蛋白创面记者。所有图像被收集在Leica SP2共聚焦显微镜，用20X物镜。箭头标示的网站伤口。在数据面板虚线S标记胚胎的轮廓。 <a href="https://www.jove.com/files/ftp_upload/50750/50750fig1highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/50750/50750fig2.jpg" width="500px" /> 图2。 弗洛-2 {KG00210}突变体丧失功能的基因突变的背景和化学显微注射胚胎。明场和受伤Flotillin-2（FLO-2）突变胚胎的荧光图像的DDC-GFP的伤口记者本土化。（A）记者扩张活动在所有表皮细胞（B）获得性功能的Flo-2（犰狳-GAL4，UAS-FLO2）突变体，抑制报告基因活性的在所有表皮细胞（C）过氧化氢 ​​（H 2 O 2的氢）注射液，扩张的代表orter活动遍及所有的表皮细胞（D）甲基-β-环糊精（MβCD）溶液显微注射，扩张的报告基因活性的贯穿所有的表皮细胞。所有图像被收集在Leica SP2共聚焦显微镜，用20X物镜。箭头标示的网站伤口。在数据面板虚线标记胚胎的轮廓。 <a href="https://www.jove.com/files/ftp_upload/50750/50750fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50750/50750fig3.jpg" width="600px" /> 图3。检测伤口反应基因的RNA转录本。 原位杂交和RNA检测在受伤的野生型胚胎荧光图像。DDC（A）和辉 （B）RNA转录积蓄在伤口部位。所有图片都是集电极特德在Leica SP2激光共聚焦显微镜，具有20X的目标。箭头标示的网站伤口。在数据面板虚线标记胚胎的轮廓。 <a href="https://www.jove.com/files/ftp_upload/50750/50750fig3highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="表1" fo:content-width="6in" src="/files/ftp_upload/50750/50750table1.jpg" width="600px" /> 表1中。表皮伤口响应记者总结。四个伤口响应记者（DDC，PLE，MSN，KKV）的插入可在第二和第三染色体上两个。在周围穿刺损伤部位在野生型背景（ 如 “本地”型）表皮细胞的数量有限所有的伤口回应记者激活荧光基因（GFP或红色荧光蛋白）。DDC和MSN伤口回应记者要求对GRH激活fluorescen的穿刺损伤（ 如 “无”表现型）后，CE基因。所有伤口记者需要的Flo-2的穿刺伤害（ 如 “全球性”表现型）后的荧光基因的定位。 <a href="https://www.jove.com/files/ftp_upload/50750/50750table1highres.jpg" target="_blank">点击这里查看大图</a> 。

Watch this Scientific Journal Video about Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos. at JoVE.com

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

极后期果蝇胚胎的胚胎表皮提供的体内系统的快速穿刺伤口响应分析，可以用基因操作或化学显微注射治疗组合以推进研究在伤口愈合翻译成哺乳动物模型。

创伤显微注射和分析<em&gt;在体内</em&gt;表皮的伤口本地化响应记者在<em&gt;果蝇</em&gt;胚胎。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

microinjection-wound-assay-vivo-localization-epidermal-wound-response

Research

JoVE Journal

Bioengineering

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

12.3K 次观看.  The City College of New York. 极后期果蝇胚胎的胚胎表皮提供的体内系统的快速穿刺伤口响应分析，可以用基因操作或化学显微注射治疗组合以推进研究在伤口愈合翻译成哺乳动物模型。 

视频: Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. Cardiovascular physiological phenotyping of the mouse heart can be easily done using fluorescence imaging employing various probes for transmembrane potential (Vm), calcium transients (CaT), and other parameters. Excitation-contraction coupling is characterized by action potential and intracellular calcium dynamics; therefore, it is critically important to map both Vm and CaT simultaneously from the same location on the heart1-4. Simultaneous optical mapping from Langendorff perfused mouse hearts has the potential to elucidate mechanisms underlying heart failure, arrhythmias, metabolic disease, and other heart diseases. Visualization of activation, conduction velocity, action potential duration, and other parameters at a myriad of sites cannot be achieved from cellular level investigation but is well solved by optical mapping1,5,6. In this paper we present the instrumentation setup and experimental conditions for simultaneous optical mapping of Vm and CaT in mouse hearts with high spatio-temporal resolution using state-of-the-art CMOS imaging technology. Consistent optical recordings obtained with this method illustrate that simultaneous optical mapping of Langendorff perfused mouse hearts is both feasible and reliable.

This paper details the dissection procedure, instrumental setup, and experimental conditions during optical mapping of transmembrane potential (Vm) and intracellular calcium transient (CaT) in intact isolated Langendorff perfused mouse hearts.

在小鼠心脏的动作电位和钙瞬变的光学测绘

The mouse heart is a popular model for cardiovascular studies due to the existence of low cost technology for genetic engineering in this species. ...

科研

生物工程

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

交付使用表皮生长因子在人胰腺癌细胞转染基因受体有针对性的明胶基于工程Nanovectors

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

简单的微流控芯片的<em在体内</em&gt;成像<em&gt; C.线虫</em&gt;<em&gt;果蝇</em&gt;和斑马鱼

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

制作定量，皮肤伤口管理一个形皮肤般的电子系统与表征

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

研究表皮生长因子受体的完整细胞使用相关显微镜的化学计量

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

对西方玉米 Rootworm、 Diabrotica virgifera virgifera、生殖转化胚胎或 CRISPR/Cas9 基因组编辑的显微注射

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

用微计算机断层扫描法对无节段3D 组织学进行固定染色、全尺寸、毫米度试样的刚性嵌入

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...