Name: identification and dissection of diverse mouse adipose depots
Uploaded: 2019-07-11T12:30:00
Description: Watch this Scientific Journal Video about Identification and Dissection of Diverse Mouse Adipose Depots at JoVE.com

O.A.M. 由 NIH 授予 DK062876 和 DK092759 支持;D.P.B. 由密歇根大学医学科学家培训计划 (T32GM007863)、密歇根大学组织培养培训项目 (T32HD007605)、密歇根大学 Rackham 优秀奖学金和泰诺未来护理奖学金提供支持。

所有动物程序均经密歇根大学机构动物护理和使用委员会(IACUC)批准。
1. 安乐死
注:在本视频协议中,使用 4 到 6 个月大的 C57BL/6J 小鼠。
<ol><li>将鼠标放入一个异常蒸汽化器室中,并将异常气流速率调整到 5% 或更大。继续接触离尼,直到呼吸停止一分钟后。然后,将鼠标从蒸发器室中取出,并使用经批准的辅助措施确认安乐死。 注:?...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dermal+Adipocytes:+From+Irrelevance+to+Metabolic+Targets?.">Dermal Adipocytes: From Irrelevance to Metabolic Targets?.</a> Trends in Endocrinology &amp; Metabolism. 27 (1), 1-10 (2016).</li><li>Iacobellis, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+systemic+effects+of+the+multifaceted+epicardial+adipose+tissue+depot.">Local and systemic effects of the multifaceted epicardial adipose tissue depot.</a> Nature Reviews Endocrinology. 11 (6), 363-371 (2015).</li><li>Addison, O., Marcus, R. L., LaStayo, P. C., Ryan, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermuscular+Fat:+A+Review+of+the+Consequences+and+Causes.">Intermuscular Fat: A Review of the Consequences and Causes.</a> International Journal of Endocrinology. 2014, 1-11 (2014).</li><li>Pond, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue+and+the+immune+system.">Adipose tissue and the immune system.</a> Prostaglandins, Leukotrienes, and Essential Fatty Acids. 73 (1), 17-30 (2005).</li><li>Kloppenburg, A. I. -. F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+emerging+player+in+knee+osteoarthritis:+the+infrapatellar+fat+pad.">An emerging player in knee osteoarthritis: the infrapatellar fat pad.</a> Arthritis Research &amp; Therapy. 15 (225), 1-9 (2013).</li><li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization,+Identification,+and+Excision+of+Murine+Adipose+Depots.">Localization, Identification, and Excision of Murine Adipose Depots.</a> Journal of Visualized Experiments. (94), e52174 (2014).</li><li>Casteilla, 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 in Molecular Biology. 155, 1-22 (2008).</li><li>de Jong, J. M., Larsson, O., Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stringent+validation+of+mouse+adipose+tissue+identity+markers.">A stringent validation of mouse adipose tissue identity markers.</a> American Journal of Physiology-Endocrinology and Metabolism. 308 (12), E1085-E1105 (2015).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ.">The adipose organ.</a> Prostaglandins, Leukotrienes, and Essential Fatty Acids. 73 (1), 9-15 (2005).</li><li>Parlee, S. D., Lentz, S. I., Mori, H., MacDougald, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+size+and+number+of+adipocytes+in+adipose+tissue.">Quantifying size and number of adipocytes in adipose tissue.</a> Methods in Enzymology. 537, 93-122 (2014).</li><li>Scheller, E. L., 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=Region-specific+variation+in+the+properties+of+skeletal+adipocytes+reveals+regulated+and+constitutive+marrow+adipose+tissues.">Region-specific variation in the properties of skeletal adipocytes reveals regulated and constitutive marrow adipose tissues.</a> Nature Communications. 6, 7808 (2015).</li><li>Scheller, E. L., 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=Use+of+osmium+tetroxide+staining+with+microcomputerized+tomography+to+visualize+and+quantify+bone+marrow+adipose+tissue+in+vivo.">Use of osmium tetroxide staining with microcomputerized tomography to visualize and quantify bone marrow adipose tissue in vivo.</a> Methods in Enzymology. 537, 123-139 (2014).</li><li>Lukjanenko, L., Brachat, S., Pierrel, E., Lach-Trifilieff, E., Feige, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+reveals+that+transient+adipogenic+activation+is+a+hallmark+of+mouse+models+of+skeletal+muscle+regeneration.">Genomic profiling reveals that transient adipogenic activation is a hallmark of mouse models of skeletal muscle regeneration.</a> PLoS One. 8 (8), e71084 (2013).</li><li>Pagano, A. F., 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=Muscle+Regeneration+with+Intermuscular+Adipose+Tissue+(IMAT)+Accumulation+Is+Modulated+by+Mechanical+Constraints.">Muscle Regeneration with Intermuscular Adipose Tissue (IMAT) Accumulation Is Modulated by Mechanical Constraints.</a> PLoS One. 10 (12), e0144230 (2015).</li><li>Khan, I. M., 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=Intermuscular+and+perimuscular+fat+expansion+in+obesity+correlates+with+skeletal+muscle+T+cell+and+macrophage+infiltration+and+insulin+resistance.">Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration and insulin resistance.</a> International Journal of Obesity. 39 (11), 1607-1618 (2015).</li><li>Sulston, R. 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随着离散化细胞簇的分子和功能特征的重要性日益得到人们所认识到,该领域的调查人员必须统一识别和切除脂肪库,以便进行进一步分析。迄今为止,对于标准化定位和隔离各种小鼠脂肪库的协议很少。以前公布的方法主要集中于一两个仓库,缺乏由不同的调查员统一识别和切除所需的细节18,19。本手稿对脂肪生物学领域是一种新颖而重要的贡献,因为它为?...

脂肪组织在全身平衡中起着关键作用,包括根据当地和全球需求储存和释放能量,热调节和分泌阿迪波金来调节能量平衡、新陈代谢和免疫反应1,2.脂肪细胞分布在整个身体离散的仓库中,在某些情况下在其微环境中担任专门角色 3、4、5。从历史上看,脂肪组织的研究集中在白色脂肪组织(WAT)及其在维持能量平衡中的作用。大多数脂肪细胞分布在整个身体在皮下和内脏WAT仓库。这些仓库的特性对于代谢疾病的差异易感性非常重要。皮下脂肪细胞,位于皮肤下面,与保护性代谢作用5相关。内脏肾上腺细胞,围绕重要器官,并包含在性腺、肾上腺、腹食、腹骨和腹环细胞内,通常与代谢紊乱有关,包括2型糖尿病和心血管疾病2.棕色脂肪组织(BAT)也得到了广泛的研究。棕色和棕色的脂肪细胞表达脱钩蛋白1(UCP1),在自适应热发生和葡萄糖平衡6,7中发挥重要作用。经典棕色的脂肪细胞包含在间体BAT仓库8。棕色腹腔细胞的簇也发现在其他位置,包括腹腔,腹腔,腹腔/腹腔,宫颈,腹腔和腹腔前科8,9。
除了它们在主要WAT和BAT仓库中的位置外,在身体4的离散利基中,还有脂肪细胞,它们可能在各自的微环境中执行特殊功能。例如,骨髓脂肪组织(BMAT)作为脂质储存体,是循环腺炎的主要来源,并与成骨细胞、成骨细胞和造血细胞10、11密切相互作用。皮肤脂肪细胞有助于广泛的过程,包括伤口愈合,免疫反应,热调节,和毛囊生长12,13。此外,表皮性腺细胞可能产生几种腺基质和化学因子,对冠状动脉疾病14的发展和进展产生局部和系统的影响。肌肉间/肌肉间WAT的扩展与增加的脂肪性、全身性胰岛素抵抗、肌肉力量和行动能力下降呈正相关。此外,在感染16期间,popliteal脂肪细胞作为淋巴扩张的脂质储存库。虽然不同关节库的具体作用一般不为人所知,但膝关节内的霍法仓库(红外线)现在被认为会导致疾病,包括前膝关节疼痛和骨关节炎17。
虽然区域差异在脂肪性质和功能正在紧张地研究,但目前由于缺乏用于识别和解剖不同小鼠库的标准化协议,该领域受到限制。以前公布的方法通常描述一个或两个特定仓库的隔离,并且缺乏统一切除18、19所需的详细程度。本手稿中描述的协议为许多不同的小鼠脂肪库的特定解剖位置和隔离步骤提供了全面的指南。虽然WAT仓库是本手稿的主要焦点,但间膜BAT的切除也作了详细的描述。使用该协议切除的脂肪组织可用于各种实验端点,包括外植研究、组织学和基因表达分析。
本手稿的目的是为调查人员提供详细的协议,以清晰、准确地识别和隔离突出和较少研究的老鼠脂肪库(图1)。这一资源将有助于更全面地研究不同利基内脂肪细胞的发育、分子和功能特征。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59499/59499fig1.jpg"> 图1:本协议对鼠标脂肪库的原理描述。这张图片改编自Bagchi等人,2018年4。<a href="https://www.jove.com/files/ftp_upload/59499/59499fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

<table>
	<tbody>
		<tr>
			<td>10% neutral buffered formalin</td>
			<td>Fisher Scientific</td>
			<td>22-110-869</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates, untreated</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3738</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol (dilute from 95%)</td>
			<td>Fisher Scientific</td>
			<td>04-355-226</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting forceps with curved tips</td>
			<td>VWR</td>
			<td>89259-946</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting pan</td>
			<td>Carolina Biological Supply Company</td>
			<td>629004</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissors (sharp/blunt tip)</td>
			<td>VWR</td>
			<td>82027-588</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze sponges</td>
			<td>Vitality Medical</td>
			<td>2634</td>
			<td>Curity 4 x 4 inch gauze sponge, 12 ply</td>
		</tr>
		<tr>
			<td>Handi-Pins for dissection</td>
			<td>Carolina Biological Supply Company</td>
			<td>629132</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors (straight)</td>
			<td>VWR</td>
			<td>470018-890</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>VWR</td>
			<td>100499-578</td>
			<td>Feather scalpel handle with blade, disposable</td>
		</tr>
	</tbody>
</table>

identification and dissection of diverse mouse adipose depots

脂肪组织是具有广泛功能的复杂器官,包括根据当地和全球需求储存和调动能量,将代谢分离以产生热量,以及分泌脂肪以调节全身平衡和免疫反应。新兴研究正在识别位于整个身体离散库的脂肪细胞发育、分子和功能特征的重要区域差异。仓库的不同属性在医学上是相关的,因为代谢疾病通常表现出油库特定的影响。该协议将为研究者提供详细的解剖图集和解剖指南,以便对各种小鼠脂肪组织进行可重复和准确的识别和切除。离散脂肪库的标准化解剖将允许详细比较其分子和代谢特性,以及在各种营养和环境条件下对局部和系统病理状态的贡献。

使用上述协议,可以成功识别和隔离各种小鼠脂肪库。皮下(A、E-F)、棕色(B)、内脏(C、D、G-J)和浮皮(K)库的解剖位置如图2所示。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59499/59499fig2.jpg"> 图2:小鼠脂肪库的解剖位置。(A) 前皮下解剖学, (B) 棕色, (C) 表皮...

Watch this Scientific Journal Video about Identification and Dissection of Diverse Mouse Adipose Depots at JoVE.com

Identification and Dissection of Diverse Mouse Adipose Depots

蛋白细胞存在于离散的仓库中,在其独特的微环境中具有不同的角色。由于发现在脂肪细胞特性和功能方面的区域差异,对仓库的标准化识别和隔离对于该领域的发展至关重要。在这里,我们提出了一个详细的协议,为各种小鼠脂肪库的切除。

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, ...

确定和解剖全身脂肪库的标准化方法对于研究分立解剖利基中脂肪细胞特性和功能的差异至关重要。该技术的主要优点是，它是一种可靠和准确的方法来识别和排泄常见和较少研究的啮齿动物脂肪库。为了识别和隔离间膜面，使用虹膜剪刀沿安乐死小鼠的皮下组织底部边缘水平切割，然后沿着仓库的自然边界进行切割。沿着组织的自然边界沿着仓库的横向边缘进行两个垂直切口。使用钳子小心地翻转仓库，以显示嵌入在白色脂肪组织中的蝴蝶形的间皮棕色脂肪组织。然后仔细解剖 BAT 远离周围的 WAT 。将脂肪组织存放在适当的容器中，用于下游分析。为了识别和隔离皮后 WAT，请将鼠标放入超供位置，并固定动物的四肢。使用钳子抬起胸骨底部的皮肤，并做一毫米的皮肤切口。将虹膜剪刀的一叶插入切口，从胸骨底部开始，到尾部底部，进行四到五厘米的中线皮肤切口。接下来，在初始垂直切口顶部进行两个一厘米水平切口，从中线横向延伸。使用钳子小心地从腹腔剥回皮肤，使用腿部定位后皮下WAT，该 WAT 应与皮肤保持关联。固定伸出的皮肤，并完全切除三角形 inguinal WAT 仓库从后肢通风口穿过腹股沟的基础。对于内脏 WAT 仓库的识别和隔离，使用钳子抬起胸骨底部的薄腹腔壁，并在帐篷组织内进行一毫米切口。将虹膜剪刀的一叶插入切口，从腹腔顶部到直肠进行四到五厘米的垂直切口。在垂直切口的顶部和底部进行两个一厘米水平切口，从中线横向延伸，并使用钳子剥回腹腔，以暴露腹腔内侧。然后将伸出的腹膜固定在解剖盘上，以收获腹腔内所含的 WAT 仓库。为了识别和隔离内脏戈纳达尔WAT，找到性腺，并使用钳子抬起相关的戈纳达尔WAT，然后使用虹膜剪刀小心地从性腺切除WAT。要切除肾库，定位肾脏并使用钳子提升肾组织。将肾脏拉到中线，看到肾和肾上腺库之间的明确划分，并切除与肾脏直接相关的 WAT。然后从 WAT 中去除位于肾脏上方的肾上腺。对于逆转性体 WAT 识别和隔离，请再次将肾脏抬到中线，并使用虹膜剪刀从后侧壁仔细解剖逆膜 WAT。要隔离波莱特 WAT，请将 patella 对解剖盘进行护理，注意膝盖后处的波莱特叶朝上。使用虹膜剪刀小心地将皮肤从后肢底部移除到脚部。使用虹膜剪刀在胃肌的中层和横向头部的劣质边框进行切割，使用钳子抬起肌肉，露出三角形的罂粟库。然后使用虹膜剪刀沿自然组织边界切除仓库。在这里，显示了小鼠皮下、棕色、内脏和波普利特仓库的解剖总位置。皮下、棕色、内脏、波普利特、组成和调节骨髓脂肪、肌肉内和骨髓脂肪库的组织学特征可以通过标准血氧素和Eosin染色方案进行评估。使用该协议分离的脂肪组织可用于各种实验终点，包括组织学分析、分子研究以及基因和蛋白质表达分析。对各种脂肪库的鉴定进行实地标准化无疑将帮助我们进一步了解它们的分子和代谢特征，以及它们对局部和系统病理状态的不同贡献。

Research

JoVE Journal

Biology

Dissection of 6.5 dpc Mouse Embryos

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

科研

生物学

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Preparation of Adipose Progenitor Cells from Mouse Epididymal Adipose Tissues

从小鼠表皮脂肪脂肪组织中制备脂肪祖细胞

Obesity and metabolic disorders such as diabetes, heart disease, and cancer, are all associated with dramatic adipose tissue remodeling. Tissue-resident ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

实验性自身免疫性脑脊髓炎的诱导及多样评价指标

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

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

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 ...

Adipose Tissue-Derived Stem Cell Isolation and Characterization

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats (Video) | JoVE 

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...