The authors have no acknowledgements.

注:所有动物的程序都与辛辛那提大学的机构动物护理和使用委员会（IACUC），并按照该指南实验动物的护理和使用来自美国国立卫生研究院（NIH出版号的审批进行，85-23，1996年修订）。 1.安乐死和消毒鼠标<ol><li>鼠标放置在含有异氟醚的supratheraputic剂量的Dropbox，并允许吸气效果。一旦老鼠被安乐死，从包装盒中取出。 </li><li>与颈部脱臼安乐死的第二个手段。 </li><li>由清洁的动物用70％乙醇消毒鼠标的外表面上。 </li></ol> 2.识别和三种不同脂肪仓库隔离<ol><li>棕色脂肪组织（BAT）隔离: <ol><li>确保毛皮是由酒精清洗湿通过表皮和真皮Laye的削减，以帮助RS。 </li><li>将鼠标在其对表背仰卧位。 </li><li>抢皮肤正下方用钳子，电梯隔膜和切割用剪刀。 </li><li>切割横向围绕鼠标的圆周以露出腹膜。 </li><li>由脱套鼠标的上半部分显示蝴蝶形的BAT仓库。保持较低的附属物和腹部，一手提拉肌肤，迈向头。 </li><li>定向鼠标，以便它被定位俯卧在桌子上。要小心，不要污染暴露车厂的头发。 </li><li>洁净手术器械和改变一双崭新的手套。 </li><li>找到肩胛和相应的库。小心取出任何肤浅的白色脂肪之上蝴蝶，然后解剖的肩胛间棕色脂肪的蝴蝶。小心避免与棕色脂肪密切相关的肌肉。 注意:使用解剖显微镜，建议去除W的海特脂肪，以及在从肩胛褐色脂肪的分离。 </li><li>去除脂肪库，并转移到2 ml离心管。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </li></ol></li><li>皮下脂肪组织（SQ）的隔离，一个白色脂肪组织得宝（WAT）: <ol><li>穿上一双崭新的手套作为脂肪仓库之间没有交叉不纯物。 </li><li>露出腹股沟，三角SQ库由去手套革底部鼠标的一半。用于保持上附肢和胸部，一手拉动皮肤向下朝向脚用另一只手。 </li><li>定位鼠标在仰卧位时注意不要污染暴露车厂的头发。 </li><li>清洁surgica升仪器和改变一双崭新的手套。 </li><li>仔细剖析的SQ脂肪的三角形。要小心，不要污染样品肌肉，脂肪的邻国，乳腺或切割任何船只和污染的血液样本。 注意:使用解剖显微镜，建议如果边界不清晰。 </li><li>去除脂肪仓库和转让〜2毫升离心管。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。 </li><li>对于染色，固定或嵌入在十月如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </li></ol></li><li>隔离性腺脂肪内脏脂肪组织（VAT）和白色脂肪组织（WAT）车厂: <ol><li>穿上一双崭新的手套作为脂肪仓库之间没有交叉不纯物。 </li><li>脊髓损伤ssors，切开腹膜横向，正下方​​的隔膜。从隔膜到直肠中间剪开腹膜冠，露出腹部器官。 </li><li>定位睾丸或卵巢，并确定所连接的白色脂肪组织，称为附睾脂肪在雄性或性腺脂肪在女性。 </li><li>洁净手术器械和改变一双崭新的手套</li><li>仔细剖析从睾丸，附睾和输精管附睾两种脂肪库。或者，如果女性，小心地从卵巢解剖两种性腺脂肪垫。 </li><li>去除脂肪库，并将它们传送至2 ml离心管中。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。 </li><li>对于染色，固定或嵌入在十月如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 </LI&gt; </ol></li></ol> 3.隔离血管周围脂肪组织（PVAT） <ol><li>孤立的心: <ol><li>莱鼠标在与向外扩展上下附属物仰卧位。 </li><li>安全附属物使用医用胶带。 </li><li>之后将鼠标上面列出，通过在剑突与钳提起制造紧张。水平切割通过隔膜，暴露胸腔的下部。 </li><li>同时维持张力，通过在剑突抬起，通过胸腔切开优朝向头部，只是到胸骨的一侧。 </li><li>拿起左肋只是次于锁骨，并沿着锁骨下长度切出朝向两个方向的腋下。胸腔和它的内容（心脏，肺等 ）现在应该清楚可见。 </li><li>通过使用无菌GA清洁胸外来​​血液和液体腔UZE吸收流体。如果收集器官或容器一定要喷出大量（补充信息）。 </li><li>一旦该区域被清除的液体，切支气管和血管连接以消除肺部，从而更好的暴露的心脏。 </li></ol></li><li>本地化和主动脉切除血管周围脂肪组织（PVAT）: <ol><li>删除以下机关更好地识别主动脉的下部分:肝，胃，脾，胰，肠和结肠癌。 </li><li>首先从识别胃和食管。切开食管的胃食管结合部，以释放从身体的腹部。 </li><li>其次，确定肠子和周围的肠系膜。切割表面通过肠系膜，因为它位于非常接近主动脉的肾部分，然后"运行肠"。 </li><li>切结肠释放尽量靠近直肠越好。因此，释放的胃，肠和结肠的鼠标。 </li><li>回复通过在附着肠系膜血管和切割移动胃，肠，结肠，胰腺和脾脏。胰腺和脾脏应该是免费的胃，小肠和结肠。 </li><li>通过削减通过肝静脉和肠系膜连接取出肝脏，删除所有叶。 </li><li>剪去内脏层和脂肪包围的肾脏。离开附加到主动脉体内肾脏作为地理标记主动脉的不同部分。 </li><li>冲洗用无菌1X PBS面积和吸收删除所有流体用无菌纱布。 </li><li>用微型剪刀和微型镊子，从它的背侧连接到脊椎和其腹侧附着到食道分离主动脉。 </li><li>按照和分离主动脉降主动脉的长度从原点的心脏在髂骨地区分叉隔离主动脉。 </li><li>识别并隔离锁骨下血管。隔离这些船只从脖子到主动脉根部，以更好地暴露在心脏主动脉结和根。 </li><li>取出胸腺然后切头臂动脉，左颈总动脉和左锁骨下动脉，使心脏的运动。 </li><li>用解剖显微镜的帮助下，查看围绕主动脉的血管周围脂肪组织（PVAT）层。 </li><li>采取非常谨慎，不捏或挤压PVAT，轻轻一拉PVAT方式，从微型钳主动脉。轻轻切PVAT的附接至与起始于胸部区域刚好优于其中隔膜位于微型剪刀主动脉。 注:解剖镜下建议。 </li><li>重复此过程主动脉的整个长度，在下腹主动脉区域，它位于正好优于主动脉血管的髂分叉精加工。 </li><li>如果主动脉弓PVAT需要，用同样的方法从较小的c取出PVATurvature拱。 </li><li>将PVAT样品分为2 ml离心管（S）。 </li><li>如果RNA或蛋白质要被提取出来，冻结由浸没在液氮中，并存储所述组织在-80℃下。冻结的样品时，为了防止恶化。如果培养，涵盖DMEF-12组织和冰上设置，直到所有的样品采集文化（补充信息）。 注意:如果有兴趣全面脂肪车厂分析更多的脂肪车厂考虑包括:腹膜后，肠系膜，大网膜，心包及腘。 </li></ol></li></ol>

<ol>
	<li>Ogden, C. L., Carroll, M. D., Kit, B. K., Flegal, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+obesity+in+the+United+States,+2009-2010.">Prevalence of obesity in the United States, 2009-2010.</a> NCHS Data Brief. 82, 1-8 (2012).</li><li>Flegal, K. M., Carroll, M. D., Kit, B. K., Ogden, 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=Prevalence+of+obesity+and+trends+in+the+distribution+of+body+mass+index+among+US+adults,+1999-2010.">Prevalence of obesity and trends in the distribution of body mass index among US adults, 1999-2010.</a> 307 (5), 491-497 (2012).</li><li>Cawley, J., Meyerhoefer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medical+care+costs+of+obesity:+an+instrumental+variables+approach.">The medical care costs of obesity: an instrumental variables approach.</a> Journal of Health Economics. 31 (1), 219-230 (2012).</li><li>Marder, W., Chang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+Obesity:+Costs,+Treatment+Patterns,+Disparities+in+Care,+and+Prevalent+Medical+Conditions.">Childhood Obesity: Costs, Treatment Patterns, Disparities in Care, and Prevalent Medical Conditions.</a> Thomson Medstat Research Brief. , (2006).</li><li>Cortez, M., Carmo, L. S., Rogero, M. M., Borelli, P., Fock, R. 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+high-fat+diet+increases+IL-1,+IL-6,+and+TNF-&#945;+production+by+increasing+NF-&#954;B+and+attenuating+PPAR-&#947;+expression+in+bone+marrow+mesenchymal+stem+cells.">A high-fat diet increases IL-1, IL-6, and TNF-&#945; production by increasing NF-&#954;B and attenuating PPAR-&#947; expression in bone marrow mesenchymal stem cells.</a> Inflammation. 36 (2), 379-386 (2013).</li><li>Sanchez-Gurmaches, J., Hung, C. M., Sparks, C. A., Tang, Y., Li, H., Guertin, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+loss+in+the+Myf5+lineage+redistributes+body+fat+and+reveals+subsets+of+white+adipocytes+that+arise+from+Myf5+precursors.">PTEN loss in the Myf5 lineage redistributes body fat and reveals subsets of white adipocytes that arise from Myf5 precursors.</a> Cell Metab. 16 (3), 348-362 (2012).</li><li>Sethi, J. K., Vidal-Puig, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thematic+review+series:+adipocyte+biology.+Adipose+tissue+function+and+plasticity+orchestrate+nutritional+adaptation.">Thematic review series: adipocyte biology. Adipose tissue function and plasticity orchestrate nutritional adaptation.</a> J Lipid Res. 48 (6), 1253-1262 (2007).</li><li>Aarsland, A., Chinkes, D., Wolfe, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+and+whole-body+fat+synthesis+in+humans+during+carbohydrate+overfeeding.">Hepatic and whole-body fat synthesis in humans during carbohydrate overfeeding.</a> Am J Clin Nutr. 65 (6), 1774-1782 (1997).</li><li>Park, A., Kim, W. K., Bae, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+of+white,+beige+and+brown+adipocytes+derived+from+mesenchymal+stem+cells.">Distinction of white, beige and brown adipocytes derived from mesenchymal stem cells.</a> World J. Stem Cells. 6 (1), 33-42 (2014).</li><li>Wu, J., 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=Beige+adipocytes+are+a+distinct+type+of+thermogenic+fat+cell+in+mouse+and.">Beige adipocytes are a distinct type of thermogenic fat cell in mouse and.</a> 150 (2), 366-376 (2012).</li><li>Giralt, M., Villarroya, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=White,+brown,+beige/brite:+different+adipose+cells+for+different+functions.">White, brown, beige/brite: different adipose cells for different functions.</a> Endocrinology. 154 (9), 2992-3000 (2013).</li><li>Casteilla, L., P&#233;nicaud, L., Cousin, B., Calise, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choosing+an+adipose+tissue+depot+for+sampling:+factors+in+selection+and+depot+specificity.">Choosing an adipose tissue depot for sampling: factors in selection and depot specificity.</a> Methods Mol. Biol. 456, 23-38 (2008).</li><li>Grant, R., Youm, Y. H., Ravussin, A., Dixit, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+adipose+tissue+leukocytosis+in+obesity.">Quantification of adipose tissue leukocytosis in obesity.</a> Methods Mol. Biol. 1040, 195-209 (2013).</li></ol>

The authors declare that they have no competing financial interests or other conflicts of interest.

肥胖可能导致大的宿主疾病发病率和该脂肪起着尚不完全清楚的作用的充分理解;因此继续研究在脂肪的字段是必要的。动物模型，特别是小鼠模型非常适合在疾病和潜在的药物治疗试验的进展最初的研究。在使用这些模型，精确的隔离和脂肪车厂切除脂肪影响了疾病的病理研究中的一个非常重要和必要的工具。 在关于脂肪库目前的文献，有文献关于代谢行为和脂肪仓库之间?...

脂肪做了一个显着的出现在媒体的聚光灯下，由于在过去几十年的20世纪肥胖的急剧增加。目前，肥胖的影响超过三分之一的成年人和在美国（美国）1儿童和青少年的17％。跨越各族，周围肥胖的统计研究表明，非西班牙裔黑人有肥胖（49.5％）的最高年龄调整率与墨西哥裔美国人（40.4％），所有西班牙裔（39.1％），和非比较西班牙裔白人（34.3％），2。肥胖的经济效应也为医疗保健系统日益受到关注。在2012年，据估计，每年的医疗照顾，肥胖在美国，2005年费用为190.2十亿，近21％的整体医疗支出预算。可悲的是，儿童肥胖，估计是负责单独$ 14十亿的直接医疗费用。在统计上，它被确定的平均医疗费用肥胖人士是2741美元高于去年比没有这种发病率3-5。 肥胖是用于各种诸如条件的主要危险因素:2型糖尿病，血脂异常，心血管疾病，癌症，肌肉骨骼疾病和慢性炎症。肥胖是深深依赖于代谢综合征等慢性疾病6-8的发病机制。随着肥胖等合并症之间的这种激烈的负极连接，科研集中注意力，以便更好地了解当前的流行和脂肪所扮演的多样和重要的角色。 从历史上看，脂肪组织被认为是无关紧要的，并仅仅看作是一个简单的填充组织。目前，脂肪已被证明在体内的功能中发挥许多重要作用:代谢，激素调节，炎症，保护和绝缘9。脂肪组织主要由脂肪细胞还含有周细胞，血管内皮细胞，单核细胞，巨噬细胞和多能干细胞8。脂肪组织是分布在整个身体中不同的仓库。主体仓库可以皮下发现，皮下，肌肉内，和本能10。脂肪库已被证明有仓库特定的代谢概况，从而衍生出一个仓库特定易感性肥胖及相关病症8。 传统上，脂肪组织已被分为两种主要类型:白色脂肪组织（WAT）和棕色脂肪组织（BAT）;虽然最近的文献表明，第三组的派驻命名BRITE或米色脂肪11。脂肪组织已被证明具有不同的颜色，形态，代谢功能，生化特征和表达10的遗传模式。在WAT脂肪细胞有一个单一的，大的脂滴和线粒体的数量可变。 WAT在身体的皮下和内脏地方是显性找到。 WAT功能主要是作为能量储存和器官保护的部位。在BAT脂肪细胞有一个多房形态和丰富的线粒体。 BAT位于主要在颈部和胸部的大血管，以及肩胛12。 BAT主要功能是调节产热7能源花费的行为。 BRITE或米色脂肪已经显示的，共享一个类似的形态和表达BAT但已被发现是源自白色脂肪细胞11。 在这个手稿中描述的手术方法为研究人员提供的能力，分析不同效果的因素，如:环境，医药，和遗传学，对脂肪;以及有益或有害作用的脂肪起着病和总体健康状况。此外，提供了一种方法来鉴定和分离不同类型的脂肪组织，以以便更好地了解车厂之间的生化关系和差异。这可以在确定身体内的位置，功能，和类型的脂肪之间的关系提供帮助。该所述方法通过提供用于毛可视化，基因表达分析，蛋白质表达分析，组织学检查，以及初级细胞系的隔离体外研究实现这一目的。目前，有许多文章，提供深入了解不同脂肪库，以及它们的解剖位置的代谢行为;但不提供一个深入的方法，对如何具体定位，识别和隔离这些仓库。这种手术方法提供了精确的技术，其允许多个站的具有夹层和污染的最小量相比较，以设计用于一个或两个仓库13-14的分离方法更为隔离。 该协议的目的是提供精确的方法对不同类型的多个解剖位置脂肪库的鉴定和分离。

<table><tbody><tr><td>Isoflurane</td><td>Med-Vet International</td><td>#RXISO-250</td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher</td><td>07-678-001</td><td></td></tr><tr><td>DMEF-12</td><td>Sigma Aldrich</td><td>D-6421</td><td>Warm in waterbath before putting on tissue.</td></tr><tr><td>2 ml Microcentrifuge tubes</td><td>Midsci</td><td>MCT-200-C-S</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma Aldrich</td><td>P5368-10PAK</td><td></td></tr></tbody></table>

localization, identification, and excision of murine adipose depots

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 reached a staggering sum of $190.2 billion in direct medical costs alone. Obesity is a major risk factor for a wide host of diseases. Historically, little was known regarding adipose and its major and essential functions in the body. Brown and white adipose are the two main types of adipose but current literature has identified a new type of fat called brite or beige adipose. Research has shown that adipose depots have specific metabolic profiles and certain depots allow for a propensity for obesity and other related disorders. The goal of this protocol is to provide researchers the capacity to identify and excise adipose depots that will allow for the analysis of different factorial effects on adipose; as well as the beneficial or detrimental role adipose plays in disease and overall health. Isolation and excision of adipose depots allows investigators to look at gross morphological changes as well as histological changes. The adipose isolated can also be used for molecular studies to evaluate transcriptional and translational change or for in vitro experimentation to discover targets of interest and mechanisms of action. This technique is superior to other published techniques due to the design allowing for isolation of multiple depots with simplicity and minimal contamination.

识别和腹股沟皮下脂肪，肩胛间褐色脂肪，内脏附睾脂肪（ 图1），以及主动脉弓血管周围脂肪，胸主动脉脂肪，肾上主动脉脂肪和下腹主动脉脂肪的本地化（ 图2），使用取得了成功所描述的手术方法。 BAT和WAT样品之间的组织学检查和分化采用HE染色（H＆E）染色（ 图3）进行了积极评价。脂联素（ADIPOQ），过氧化物酶体增殖物激活受体γ（PPAR-γ），的RNA水?...

Watch this Scientific Journal Video about Localization, Identification, and Excision of Murine Adipose Depots at JoVE.com

Localization, Identification, and Excision of Murine Adipose Depots

由于肥胖等合并症之间的激烈负极连接，对脂肪的作用发挥的研究在疾病和整体健康是必要的。我们提出了一个协议，用于分离和脂肪仓库允许利用原位脂肪研究的切除和体外方法。

本地化识别，切除脂肪小鼠油库，并

Obesity has increased dramatically in the last few decades and affects over one third of the adult US population. The economic effect of obesity in 2005 ...

localization-identification-and-excision-of-murine-adipose-depots

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

40.9K 次观看.  University of Cincinnati College of Medicine. 由于肥胖等合并症之间的激烈负极连接，对脂肪的作用发挥的研究在疾病和整体健康是必要的。我们提出了一个协议，用于分离和脂肪仓库允许利用原位脂肪研究的切除和体外方法。 

视频: Localization, Identification, and Excision of Murine Adipose Depots

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

科研

发育生物学

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...

生物工程

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

免疫与感染

Lymphatic and Blood Network Analysis During Obesity

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still not elucidated. However, obesity is characterized by impaired lymphatic function and increased vessel permeability. Inversely, lymphatic dysfunction induces obesity in mice, suggesting a significant interplay between lymphatic vessels and the adipose tissue. Therefore, understanding factors leading to lymphatic dysfunction might open new therapeutic windows to prevent obesity and associated comorbidities. The first step in this process requires a precise and detailed visualization of the lymphatic network in healthy and inflamed adipose tissue. Here, we describe a rapid, inexpensive, and efficient method that allows to label and analyze lymphatic and blood vessels. This approach takes advantage of the skin-draining brachial lymph node localization within the subcutaneous adipose tissue. The lymphatic arborization of this tissue can be revealed by injecting fluorochrome-conjugated lectins subcutaneously. Moreover, the in vivo labeling approach provides a way to evaluate lymphatic vessel density and functions. Coupled to blood vessel, adipocyte and immune cell staining, the protocol allows for high-resolution mapping of the subcutaneous adipose tissue by 3D imaging.

Obesity is a growing global public health issue. It has been previously associated with lymphatic dysfunction, suggesting a vital crosstalk between the adipose tissue and the lymphatic system. Here, we propose an accessible methodology allowing the distinct labeling of blood and lymphatic vasculatures within the subcutaneous adipose tissue.

Lymphatic collecting vessels and lymph nodes are inevitably embedded in adipose tissue. The physiological significance of this observation remains still ...

医学

Identification and Dissection of Diverse Mouse Adipose Depots

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, uncoupling of metabolism to generate heat, and secretion of adipokines to regulate whole-body homeostasis and immune responses. Emerging research is identifying important regional differences in the developmental, molecular, and functional profiles of adipocytes located in discrete depots throughout the body. Different properties of the depots are medically relevant since metabolic diseases often demonstrate depot-specific effects. This protocol will provide investigators with a detailed anatomic atlas and dissection guide for the reproducible and accurate identification and excision of diverse mouse adipose tissues. Standardized dissection of discrete adipose depots will allow detailed comparisons of their molecular and metabolic characteristics and contributions to local and systemic pathologic states under various nutritional and environmental conditions.

Adipocytes exist in discrete depots and have diverse roles within their unique microenvironments. As regional differences in adipocyte character and function are uncovered, standardized identification and isolation of depots is crucial for advancement of the field. Herein, we present a detailed protocol for the excision of various mouse adipose depots.

各种鼠标脂肪库的识别和解剖

Adipose tissues are complex organs with a wide array of functions, including storage and mobilization of energy in response to local and global needs, ...

生物学

Isolation and Identification of Vascular Endothelial Cells from Distinct Adipose Depots for Downstream Applications

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone regulation, barrier functions, and angiogenesis. Endothelial cell dysfunction is a hallmark predictor and major driver for the progression of severe cardiovascular diseases, yet the underlying mechanisms remain poorly understood. The ability to isolate and perform analyses on endothelial cells from various vascular beds in their native form will give insight into the processes of cardiovascular disease. This protocol presents the procedure for the dissection of mouse subcutaneous and mesenteric adipose tissues, followed by isolation of their respective arterial vasculature. The isolated arteries are then digested using a specific cocktail of digestive enzymes focused on liberating functionally viable endothelial cells. The digested tissue is assessed by flow cytometry analysis using CD31+/CD45&#8722; cells as markers for positive endothelial cell identification. Cells can be sorted for immediate downstream functional assays or used to generate primary cell lines. The technique of isolating and digesting arteries from different vascular beds will provide options for researchers to evaluate freshly isolated vascular cells&#160;from arteries of interest and allow them to perform a wide range of functional tests on specific cell types.

This protocol details a method for the dissection of mouse adipose depots and the isolation and digestion of respective arteries to liberate and then identify the endothelial cell population. Freshly isolated cells used in downstream applications will advance the understanding of vascular cell biology and the mechanisms of vascular dysfunction.

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

Vascular endothelial cells lining the wall of the vascular system play important roles in a variety of physiological processes, including vascular tone ...

小鼠白色脂肪组织的半自动分离

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

作者聚焦：使用组织解离剂从小鼠白色脂肪组织中半自动分离基质血管组分  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

小鼠 scBAT 的形态学和分子学特征

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily.
To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT&#39;s small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

作者聚焦：优化锁骨上棕色脂肪组织提取以进行遗传分析

Here, we provide a practical procedure for dissecting and performing histological and gene expression analyses of murine supraclavicular brown adipose tissue.

小鼠锁骨上棕色脂肪组织的解剖、组织学处理及基因表达分析

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly ...

本地化识别，切除脂肪小鼠油库，并

摘要

探索更多视频

此视频中的章节

脂肪库专用SCA1的免疫磁性分离

荧光染料标记脂质体体内组织特异性细胞摄取的制备、给药和评估

从小鼠腹内脂肪库分离成脂肪和纤维炎症基质细胞亚群

Lymphatic and Blood Network Analysis During Obesity

各种鼠标脂肪库的识别和解剖

从不同的脂肪库分离和鉴定血管内皮细胞，用于下游应用

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

使用组织解离器从小鼠白色脂肪组织中半自动分离基质血管部分

小鼠锁骨上棕色脂肪组织的解剖、组织学处理及基因表达分析