这个项目是由教育的新加坡教育部（MOE，授权号2013-T2-2-126）和健康，美国国家研究院（NIH，授予数量1R21AT008452-01A1）赠款。 TY从生物科学系新加坡国立大学获得了硕士研究生奖学金。我们感谢新加坡国立大学中心生物成像科学（CBIS）的共焦单位的一贯支持。

所有实验均按照新加坡国立大学（R14-293）批准的机构动物护理和使用委员会（IACUC）协议执行。 1.鱼饲养和胚胎的收集<ol><li>提高WT，CTSK:nlGFP 24，RANKL:HSE:CFP 24和OSX:mCherry 21单，或在26°C复合转基因鱼青鳉控制的光周期下（14小时光照，10小时黑暗）来诱导产卵。 </li><li>每日产卵的灯打开后的第一个30分钟内发生。鸡蛋丝，通过粘在一起，并连接到女性的腹部了好几个小时。用细孔网搭上了成年女性携带一个鸡蛋集群。让鱼短暂地休息了网，然后轻轻按摩鱼的腹部从女性腹部仔细剥离受精卵集群。 注:健康女性的青鳉能生产10 - 每天20个蛋大约5个月。 </li><li>将鸡蛋打入60毫米的塑料培养皿。用塑料吸管5冲洗胚胎- 0.3X Danieau 10毫升的溶液（鱼介质; 19.3毫摩尔NaCl，0.23毫摩尔的KCl，0.13毫硫酸镁 ，0.2毫米的Ca（NO 3）2，和1.7毫米的HEPES，pH值7.0）。添加1毫升的0.25％（重量/体积）亚甲蓝原液2.5升鱼介质，以防止霉菌生长。 </li><li>轻轻摇动鸡蛋集群，形成附着丝结。使用镊子小心地从受精卵集群删除附件细丝，以获得单个胚胎（ 图1A）。 </li><li>根据2004年13岩松阶段的胚胎。 </li><li>文化20 - 为60毫米的塑料培养皿30胚胎在28℃培养箱。每天更换介质，以保证胚胎的正常发育。 注:各地孵化阶段（8时 - 9天受精后，DPF）是生存尤为关键。删除自由浮动绒毛膜保持中等清洁，以确保良好的幼体成活率。 </li></ol> 2.转基因胚胎筛选<ol><li>使用配有汞灯荧光成像和GFP，RFP和CFP过滤器立体显微镜来筛选使用40X放大倍率荧光记者表达转基因胚胎。 </li><li>可视地识别OSX:mCherry胚胎由mCherry记者表达在早期形成颅骨，如cleithrum上后头部（ 图1B，箭头），和副蝶骨的两侧，在腹侧颅骨的中央位置（ 图1B中 ，箭头）。 注:记者表情从5 DPF起21开始。 </li><li>确定CTSK:nlGFP胚胎强nlGFP表达的头部（ 图1C，箭头）和尾部（ 图1C，箭头），启动6 DPF。 注:内源性破骨细胞只能形成后的21 DPF。 nlGFP表达在早期阶段（6 DPF）细胞不是破骨细胞，但另一方面，到目前为止未鉴定，CTSK阳性细胞24。 </li><li>确定RANKL:HSE:由无处不在CFP CFP表达转基因胚胎短暂热激处理20分钟，在39°C，在2 DPF或更高版本用于筛选后进行。 注:RANKL和CFP转基因是相同的双向热休克元件（HSE）的控制之下。 CFP表达式表示成功RANKL诱导24。 </li><li>在9 DPF 2小时热激处理或更高版本诱使大量异位破骨细胞在树干地区，从而导致骨质疏松症样表型24 -执行1.5。 注:在休眠破骨细胞的祖细胞，内源性没有被触发的异位激活在9 DPF结果转基因诱导RANKL表达前21 DPF。使用水浴，以获得稳定的39℃的条件。让含培养皿鳉胚胎浮在水面上。确保培养皿的盖子是干燥，防止菜沉没。 </li><li>从化合物系，例如RANKL屏幕胚胎:HSE:CFP / CTSK:nlGFP双转基因和OSX:mCherry / RANKL:HSE:CFP / CTSK:nlGFP三重转基因，根据每个单独的转基因的表达模式。 注:半合子和纯合子转基因胚胎是由记者转基因的不同荧光水平区分。纯合胚胎有荧光强度被大致相比，半合子转基因的一倍。 HSE:CFP和CTSK:这是纯合子两者RANKL复合线nlGFP被反复incrossing几代获得。对于三转基因OSX:mCherry / RANKL:HSE:CFP / CTSK:nlGFP鱼，<html>纯合子RANKL:HSE:CFP / CTSK:mCherry运营商:nlGFP鱼纯合子OSX交叉。由此产生的杂合三转基因后代中提出并incrossed获得胚胎纯合子RANKL:HSE:CFP。该RANKL:HSE:CFP转基因必须以获得异位破骨细胞的有效诱导纯合的。 </li></ol><img alt="图1" src="/files/ftp_upload/55025/55025fig1.jpg" /> 图1:WT和转基因青鳉胚胎7天受精后（DPF）。 一 。 WT胚胎明照明观察。 B点 。转基因胚胎显示OSX:各地cleithrum（箭头）和副蝶骨（箭头）mCherry表达。 ℃。转基因胚胎显示CTSK:头部（箭头）nlGFP表达和尾（箭头）。比例尺:500微米。025 / 55025fig1large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 3.鳉幼虫双膦酸盐治疗<ol><li>制备含有用于剂量 - 反应研究不同浓度的二膦酸盐（BPS）的解决方案。 注:此协议使用的示范性BP是阿仑膦酸钠。 <ol><li>溶解在鱼网上平台阿仑以100μg/ mL的浓度，以制备原料溶液。 </li><li>使用涡旋混合器以确保完全溶解。储存在4℃的储液。 </li><li>由原液鱼介质稀释到一系列浓度的准备不同的工作的解决方案（ 即 ，25％，37.5，50，62.5，和75微克/毫升）。 注意:不同的药物可以具有不同的吸收，分布，代谢和排泄（ADME）的参数，它必须在使用该鳉幼虫系统在测试过程中被考虑。此外，药物的溶解度和稳定性可能会发生变化，当施加作为水溶液。较少的水溶性化合物可能需要先溶解在有机溶剂，例如DMSO。在这种情况下，一个储备液在DMSO中，然后将其进一步在鱼培养基稀释制备。需要注意的是在水（鱼介质）工作溶液可以储存在冰箱中若干周。然而，含有DMSO溶液必须存储在RT以防止结晶。 </li></ol></li><li>鳉转移到幼虫6孔板（SIX幼虫/孔）随后BP（阿仑膦酸钠）的治疗。 </li><li>取出鱼中仔细用干净的塑料吸管，并添加阿仑膦酸钠解决每一个体积小（大约0.5毫升）好。 </li><li>避免剩鱼的介质，因为加入的BP溶液可能稀释，这是浓缩不太阿仑解决方案尤其重要。 </li><li>除去从每个孔阿仑溶液（高达0.5毫升）的小体积，用干净的塑料吸管和具有较大VOLU取代它箱（4毫升）阿仑膦酸钠溶液。 </li><li>每天换液，以保证正常的胚胎发育。 </li></ol> 4.矿化骨基质的活染<ol><li>溶解0.5克茜素配位的（ALC;茜素-3-甲基亚膦酸）或在50mL鱼介质加入0.05g钙黄绿素的制备1％和0.1％的储备溶液，分别。使用涡旋混合器以确保完全溶解。 注:不加入亚甲蓝鱼介质在此被使用和随后的步骤，以减少在幼虫自发荧光。 </li><li>使用注射器和单次使用的过滤器（0.2微米）过滤染色液。存储在黑暗中在RT下过滤溶液。 注:过滤，清晰的ALC染色溶液的颜色为暗黄色至橙色。过滤后，清钙黄绿素溶液的颜色为亮黄色。解决方案可以用于数月。 </li><li>稀释鱼介质过滤ALC或钙黄绿素原液1:10在28℃培养箱2.5小时（0.01％钙黄绿素溶液），如果使用的幼虫和17之间9 DPF - 孵育1.5鳉幼虫 - 2小时（0.1％ALC溶液）或2。保持样品在黑暗中。 </li><li>传输用干净的塑料吸管幼虫鲜鱼网上平台。 </li><li>用干净的塑料吸管取出鱼中，添加新鲜的鱼网上平台。重复此步骤3 - 4次，直到没有巴西龟或黄色染色溶液（ALC或钙黄绿素，分别）被遗留下来的。它们安装成像避免从介质表面荧光前60分钟 - 留在30鱼介质的幼虫。 注:0.1％ALC染色液是有害的青鳉幼虫延长曝光时间。长于2小时的孵育时间影响幼虫的存活。因此，浓度和染色时间需以达到最佳的胚胎存活率和染色结果的不同阶段进行优化。 </li></ol> 5.实时荧光成像</<html> p&gt; <ol><li>麻醉中鱼培养基含0.01％三卡因（3-氨基苯甲酸乙酯甲磺酸盐）的青鳉幼虫。 注:麻醉幼虫变成固定后5 - 在三卡因溶液10分钟，通常要么趴在其两侧或他们的背上。 </li><li>使用塑料微加载根据感兴趣的区域，以定向的幼虫。在这个协议中使用的幼虫的方向是横向。 </li><li>使用荧光照明的成像立体显微镜。拍摄图像时，侧重于幼虫（头，躯干前，后躯干和尾巴）的不同部分使用高倍率。在使用适当的图像处理软件（在图3G插图）重叠的区域拼接单个图像一起。 注意:这有助于提高所有相关身体部分的正确的焦平面的图像质量。 </li><li>返回幼虫鱼介质恢复后成像。 </li></ol>TLE"&gt; 6。现场共焦成像<ol><li>麻醉鱼中有0.01％三卡因幼虫5 - 10分钟，直到他们成为固定的。 </li><li>溶解通过在微波炉中加热低熔点琼脂糖在鱼类中1.5％。凉爽该溶液到约30℃。 </li><li>在鱼介质1毫升液体1.5％低熔点的琼脂糖玻璃底培养皿 - 添加0.5。转移麻醉幼虫变成用干净的塑料吸管的解决方案。 注意:要特别防范措施，液体低熔点琼脂糖的温度足够低，以不伤害幼虫。 </li><li>琼脂糖固化之前，用塑料微加载到幼虫推到培养皿的底部，并根据感兴趣的区域定向的幼虫。在这个协议中使用的幼虫的方向是横向。 注:该样品是准备共聚焦实时成像琼脂糖完全凝固后。 </li><li>使用激光共聚焦显微镜ACQuire图像。 </li><li>对于mCherry和ALC染色分析使用543纳米激光线。使用488nm的激光线nlGFP和钙黄绿素染色分析。 </li><li>成像后，鱼介质添加到培养皿，并使用一对细的注射器针头（27国祥1.5"）的仔细从琼脂糖除去幼虫。琼脂糖残余附着到培养皿鱼介质以回收转移幼虫。 </li><li>处理使用图像分析软件27的图像。 </li></ol>

<ol>
	<li>Ablain, J., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+fish+and+men:+using+zebrafish+to+fight+human+diseases.">Of fish and men: using zebrafish to fight human diseases.</a> Trends Cell Biol. 23 (12), 584-586 (2013).</li><li>Ansai, S., Kinoshita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+using+CRISPR/Cas+system+in+medaka.">Targeted mutagenesis using CRISPR/Cas system in medaka.</a> Biol Open. 3 (5), 362-371 (2014).</li><li>Apschner, A., Schulte-Merker, S., Witten, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+all+bones+are+created+equal-using+zebrafish+and+other+teleost+species+in+osteogenesis+research.">Not all bones are created equal-using zebrafish and other teleost species in osteogenesis research.</a> Methods Cell Biol. 105, 239-255 (2011).</li><li>Bajoghli, B., Aghaallaei, N., Heimbucher, T., Czerny, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+artificial+promoter+construct+for+heat-inducible+misexpression+during+fish+embryogenesis.">An artificial promoter construct for heat-inducible misexpression during fish embryogenesis.</a> Dev Biol. 271 (2), 416-430 (2004).</li><li>Barrett, R., Chappell, C., Quick, M., Fleming, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+high+content,+in+vivo+model+of+glucocorticoid-induced+osteoporosis.">A rapid, high content, in vivo model of glucocorticoid-induced osteoporosis.</a> Biotechnol J. 1 (6), 651-655 (2006).</li><li>Centanin, L., Ander, J. J., Hoeckendorf, B., Lust, K., Kellner, T., Kraemer, I., Urbany, C., Hasel, E., Harris, W. A., Simons, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exclusive+multipotency+and+preferential+asymmetric+divisions+in+post-embryonic+neural+stem+cells+of+the+fish+retina.">Exclusive multipotency and preferential asymmetric divisions in post-embryonic neural stem cells of the fish retina.</a> Development. 141 (18), 3472-3482 (2014).</li><li>Charles, J. F., Aliprantis, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts:+more+than+'bone+eaters.">Osteoclasts: more than 'bone eaters.</a> Trends Mol Med. 20 (8), 449-459 (2014).</li><li>DeLaurier, A., Eames, B. F., Blanco-Sanchez, B., Peng, G., He, X., Swartz, M. E., Ullmann, B., Westerfield, M., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+sp7:EGFP:+a+transgenic+for+studying+otic+vesicle+formation,+skeletogenesis,+and+bone+regeneration.">Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration.</a> Genesis. 48 (8), 505-511 (2010).</li><li>Du, S. J., Frenkel, V., Kindschi, G., Zohar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+normal+and+defective+bone+development+in+zebrafish+embryos+using+the+fluorescent+chromophore+calcein.">Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.</a> Dev Biol. 238 (2), 239-246 (2001).</li><li>Eriksen, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+bone+remodeling.">Cellular mechanisms of bone remodeling.</a> Rev Endocr Metab Disord. 11 (4), 219-227 (2010).</li><li>Hockendorf, B., Thumberger, T., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+embryogenesis:+a+perspective+for+light+sheet+microscopy.">Quantitative analysis of embryogenesis: a perspective for light sheet microscopy.</a> Dev Cell. 23 (6), 1111-1120 (2012).</li><li>Inohaya, K., Takano, Y., Kudo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+teleost+intervertebral+region+acts+as+a+growth+center+of+the+centrum:+in+vivo+visualization+of+osteoblasts+and+their+progenitors+in+transgenic+fish.">The teleost intervertebral region acts as a growth center of the centrum: in vivo visualization of osteoblasts and their progenitors in transgenic fish.</a> Dev Dyn. 236 (11), 3031-3046 (2007).</li><li>Iwamatsu, T. <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+normal+development+in+the+medaka+Oryzias+latipes.">Stages of normal development in the medaka Oryzias latipes.</a> Mech Dev. 121 (7), 605-618 (2004).</li><li>Kirchmaier, S., Hockendorf, B., Moller, E. K., Bornhorst, D., Spitz, F., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+site-specific+transgenesis+and+enhancer+activity+tests+in+medaka+using+PhiC31+integrase.">Efficient site-specific transgenesis and enhancer activity tests in medaka using PhiC31 integrase.</a> Development. 140 (20), 4287-4295 (2013).</li><li>Kirchmaier, S., Naruse, K., Wittbrodt, J., Loosli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+and+genetic+toolbox+of+the+teleost+medaka+(Oryzias+latipes).">The genomic and genetic toolbox of the teleost medaka (Oryzias latipes).</a> Genetics. 199 (4), 905-918 (2015).</li><li>Komori, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+osteoporosis.">Animal models for osteoporosis.</a> Eur J Pharmacol. 759, 287-294 (2015).</li><li>Mackay, E. W., Apschner, A., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bone+to+pick+with+zebrafish.">A bone to pick with zebrafish.</a> Bonekey Rep. 2, 445 (2013).</li><li>MacRae, C. A., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+tools+for+drug+discovery.">Zebrafish as tools for drug discovery.</a> Nat Rev Drug Discov. 14 (10), 721-731 (2015).</li><li>Mitchell, R. E., Huitema, L. F., Skinner, R. E., Brunt, L. H., Severn, C., Schulte-Merker, S., Hammond, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+studying+osteoarthritis+genetics+in+zebrafish.">New tools for studying osteoarthritis genetics in zebrafish.</a> Osteoarthritis Cartilage. 21 (2), 269-278 (2013).</li><li>Renn, J., Buttner, A., To, T. T., Chan, S. J., Winkler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+col10a1:nlGFP+transgenic+line+displays+putative+osteoblast+precursors+at+the+medaka+notochordal+sheath+prior+to+mineralization.">A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization.</a> Dev Biol. 381 (1), 134-143 (2013).</li><li>Renn, J., Winkler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osterix-mCherry+transgenic+medaka+for+in+vivo+imaging+of+bone+formation.">Osterix-mCherry transgenic medaka for in vivo imaging of bone formation.</a> Dev Dyn. 238 (1), 241-248 (2009).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Spoorendonk, K. M., Peterson-Maduro, J., Renn, J., Trowe, T., Kranenbarg, S., Winkler, C., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+and+Cyp26b1+are+critical+regulators+of+osteogenesis+in+the+axial+skeleton.">Retinoic acid and Cyp26b1 are critical regulators of osteogenesis in the axial skeleton.</a> Development. 135 (22), 3765-3774 (2008).</li><li>To, T. T., Witten, P. E., Renn, J., Bhattacharya, D., Huysseune, A., Winkler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rankl-induced+osteoclastogenesis+leads+to+loss+of+mineralization+in+a+medaka+osteoporosis+model.">Rankl-induced osteoclastogenesis leads to loss of mineralization in a medaka osteoporosis model.</a> Development. 139 (1), 141-150 (2012).</li><li>Wakamatsu, Y., Pristyazhnyuk, S., Kinoshita, M., Tanaka, M., Ozato, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+see-through+medaka:+a+fish+model+that+is+transparent+throughout+life.">The see-through medaka: a fish model that is transparent throughout life.</a> Proc Natl Acad Sci USA. 98 (18), 10046-10050 (2001).</li><li>Witten, P. E., Huysseune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+view+on+mechanisms+and+functions+of+skeletal+remodelling+in+teleost+fish,+with+special+emphasis+on+osteoclasts+and+their+function.">A comparative view on mechanisms and functions of skeletal remodelling in teleost fish, with special emphasis on osteoclasts and their function.</a> Biol Rev Camb Philos Soc. 84 (2), 315-346 (2009).</li><li>Yu, T., Witten, P. E., Huysseune, A., Buettner, A., To, T. T., Winkler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+osteoclast+inhibition+by+bisphosphonates+in+a+medaka+osteoporosis+model.">Live imaging of osteoclast inhibition by bisphosphonates in a medaka osteoporosis model.</a> Dis Model Mech. 9 (2), 155-163 (2016).</li></ol>

作者宣称，他们没有竞争或经济利益。

该议定书中的关键步骤至关重要的是，比较不同样品时对热休克处理的条件是一致的和稳定的。稳定的温度条件下保证RANKL诱导的相似的水平在转基因幼虫，因此，可比破骨细胞形成，其可以通过筛选CTSK予以确认:nlGFP表达。最终，这将导致类似的程度诱导异位骨吸收和骨质疏松症样病变，如通过ALC染色验证。这样的实验设计，然后允许确定和各种抗吸收药物或以不同的浓...

脊椎动物骨架提供了器官的结构支撑和保护，允许的移动性，并作为钙源。在整个生命中，细胞外骨基质连续上交保持骨的稳定性和刚性。这个过程需要紧密协调的活性和骨形成的成骨细胞和骨再吸收的破骨细胞的相互作用。成骨细胞是从多能间充质祖细胞衍生的和产生胶原，以形成类骨质，骨基质10的蛋白质的部分。成骨细胞的破骨细胞相互作用以实现这两种细胞类型，这是需要控制骨稳态7的平衡的活性。由于这些复杂的调控相互作用，药物治疗和骨稳态反应不能充分使用体外研究审查。因此，存在对动物模型的强烈需求。相比于细胞培养物的设置， 在体内模型可以提供有价值的洞察骨环境中的多细胞网络。 众多的小鼠模型，适用于各种人类骨骼疾病包括骨质疏松症16存在。但是，大小和小鼠胚胎的无障碍代表骨架过程实时成像显著局限性。小硬骨鱼，另一方面，作为体内成像一个有吸引力的替代方案。斑马鱼（ 斑马鱼 ）和青鳉（ 青鳉 ）已经成为骨骼研究流行的动物模型，在过去二十年中17，19，22，24。骨在硬骨鱼和哺乳动物是非常相似的，无论在结构和生理水平，和许多关键调节基因和信号途径的是保守3。作为哺乳动物，硬骨鱼仔细调节成骨细胞和破骨细胞，以平衡骨形成和骨吸收26的活性。最重要的是，网络连接的光学透明性sh的幼虫允许使用荧光报告标记骨细胞和钙化骨骼基质8，9，12，21，23，这有利于细胞过程在活的动物中观察。另外，已经产生了一系列的遗传工具，以促进在鱼类生物医学相关的研究。特别是对于青鳉，用于通过CRISPR / Cas9 2，细胞谱系追踪6，和转基因14最近已建立和现在都在使用15广泛点特异性靶向基因突变的方法。 小硬骨鱼类幼虫已成功地用于化工屏幕，这导致了一些药理相关药物1,18发现。 鱼幼体耐受低浓度的DMSO，并且能够吸收化合物从它们的水生环境中，无论是通过皮肤或通过胃肠道1,5。我们以前的实验室代表表达在骨细胞荧光报告各种osteoblast-和破骨细胞特异性启动子的控制下orted转基因黑点青鳉的行。这些包括过早成骨细胞（ 骨胶原10A1，COL10A1; osterix的 ，OSX）20，21，成熟的成骨细胞（ 骨钙蛋白 ，OSC）27，和破骨细胞（ 组织蛋白酶K，CTSK）24。我们还产生了一个热休克诱导型启动子24的控制下表达的破骨细胞诱导因子核因子κB配体（RANKL）的受体激活剂的转基因线。 在这个系统中RANKL诱导导致异位形成活跃的破骨细胞。这导致增加的骨吸收和严重的骨质疏松症样表型，与在椎体急剧降低矿化。我们最近发现，在此模型中的破骨细胞活性可通过二膦酸盐依替和阿仑，总重量被阻止Ø药物在人类治疗骨质疏松症常用，从而验证青鳉作为一个合适的模型系统，骨质疏松症27。 由于其大尺寸育雏，快速发展，胚胎的体积小，转基因青鳉幼虫是唯一适用于骨质疏松症药物的大规模筛选和骨细胞行为的体内分析。在青鳉研究从而可以有效地在细胞培养物和在那些旨在发现新的治疗靶点和新疗法为人类骨疾病的小鼠互补实验。 在本研究中，我们描述了一个协议，把青鳉骨记者幼虫常见的骨质疏松症药物阿仑膦酸钠。我们还详细描述了如何治疗幼虫安装和骨基质和骨细胞的实时成像准备。这些协议可以很容易地适用于其他的小的化学化合物，要么工作作为骨合成或抗骨吸收的药物。&lt;/ P&gt;

<table border="0" cellpadding="0" cellspacing="0" width="1101">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Alendronate&#160;</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:140px;">A4978</td>
			<td style="width:535px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">alizarin-3-methyliminodiacetic acid, Alizarin Complexone</td>
			<td>Sigma</td>
			<td>A3882</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcein</td>
			<td>Sigma</td>
			<td>C0875</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ImageJ (1.4.3.67)</td>
			<td style="width:177px;">National Institute of Health (NIH)</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">LSM 510 Meta confocal&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">LSM Image Browser (4.2.0.121)</td>
			<td>Zeiss</td>
			<td></td>
			<td>http://www.zeiss.com/microscopy/en_de/downloads/lsm-5-series.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micro-loader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>Eppendorf ep T.I.P.S 20 &#956;l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">NIS-Elements BR 3.0 software</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Photoshop CS6 (13.0.0.0)</td>
			<td>Adobe</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">SMZ1000 stereomicroscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

drug treatment and in vivo imaging of osteoblast-osteoclast interactions in a medaka fish osteoporosis model

骨形成的成骨细胞与骨再吸收的破骨细胞相互作用，以协调的骨基质的营业额，并控制骨骼稳态。青鳉和斑马鱼幼体被广泛用于分析骨细胞的骨形成，变性，修复期间的行为。其光学清晰度允许荧光标记的骨细胞和结合到矿化的骨基质的荧光染料的可视化。我们的实验室已产生的转基因黑点青鳉鱼表达热激诱导型启动子的控制下的破骨细胞诱导因子核因子κB配体（RANKL）的受体激活剂。在过量形成活化的破骨细胞，其可以在记者线路用nlGFP表达的组织蛋白酶K（CTSK）启动子的控制下，被可视化的RANKL结果的异位表达。 RANKL诱导和异位破骨细胞形成导致了严重的骨质疏松样的表型。复合转基因青鳉李表达CTSK内斯:nlGFP在破骨细胞，以及在过早的成骨细胞的osterix的 （OSX）启动子的控制下，mCherry，可用于研究两种细胞类型的相互作用。这有利于细胞行为骨退化和修复的条件下， 在体内观察。在这里，我们描述了使用该系统的测试在人骨质疏松症的治疗中通常使用的药物和描述了用于实时成像的协议。在青鳉模型补充在细胞培养物和小鼠中的研究，并提供一种新颖的系统，用于骨骼系统药物作用的体内分析。

丰富的产蛋数，以及幼虫的小尺寸，使青鳉药物筛选的优秀典范。单个6孔板被用来培养多达36个幼虫，这是足以提供统计学显著数据。用鱼骨骼分析的另一大优势是在做实时成像的可能性。仔鱼的透明度允许使用荧光蛋白的标记的骨细胞，以及利用结合于骨基质，以可视化矿化染料。鱼幼体易于处理，并用于成像样品制备是简单的（ 图2）。 <p class="jove_content" fo:keep-together.within-page...

Watch this Scientific Journal Video about Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model at JoVE.com

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

戒毒治疗和<em&gt;在体内</em&gt;在青鳉鱼骨质疏松模型成骨破骨细胞相互作用的成像

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

drug-treatment-vivo-imaging-osteoblast-osteoclast-interactions-medaka

Research

JoVE Journal

Developmental Biology

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

9.1K 次观看.  National University of Singapore. Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

视频: Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

造血干细胞的自动定量 - 在脱钙骨的组织学图像间质细胞的相互作用

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

转录分析的新生RNA FISH<em&gt;在体内</em&gt;滋养巨细胞或<em&gt;体外</em&gt; Ectoplacental锥外植体的短期文化

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis. The protocol presented here shows how to combine standard labeling, gain- and loss-of-function genetic or RNA interference (RNAi)-, and microscopic approaches to use this model to visually dissect and functionally analyze these processes on a molecular level. As an example of a labeling approach, the protocol outlines the generation of transgenic animals with fluorescent fusion proteins for live analysis of tubulogenesis. As an example of a genetic approach, it highlights key points of a visual RNAi-based interaction screen designed to modify a gain-of-function cystic canal phenotype. The specific methods described are how to: label and visualize the canals by expressing fluorescent proteins; construct a targeted RNAi library and strategize RNAi screening for the molecular analysis of canal morphogenesis; visually assess modifications of canal phenotypes; score them by dissecting fluorescence microscopy; characterize subcellular canal components at higher resolution by confocal microscopy; and quantify visual parameters. The approach is useful for the investigator who is interested in taking advantage of the C. elegans excretory canal for identifying and characterizing genes involved in the phylogenetically conserved processes of intracellular lumen and unicellular tube morphogenesis.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans excretory canal is a unique single-cell model for the visual in vivo analysis of de novo polarized membrane biogenesis. This protocol describes a combination of standard genetic/RNAi and imaging approaches, adaptable for the identification and characterization of molecules directing unicellular tubulogenesis, and apical membrane and lumen biogenesis.

C. 线虫排泄管作为细胞内腔形态发生和体内极化膜生物在单个细胞中的模型: GFP-融合、rna 干扰界面和成像的标记

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

有丝分裂过程中染色体分离的活体细胞成像

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...