Name: isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology
Uploaded: 2017-10-30T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology at JoVE.com

作者想承认4月诡计和 Dr. 罗杰勃洛为协助与动物尸检和腺隔离, 分别。这项工作的经费是由费城骨科医学中心的长期老化疾病的支持。

本议定书中涉及动物主体的程序由费城骨科医学院动物保育和使用委员会审查批准, 所有技术均在严格道德准则. 
 1. 示例采购和处理 <ol> <li> 选择适当的动物主题和地点到 CO 2 会议厅。对于这个实验, 使用8-10 周的老女性产妇 B6。Cg-Cav1tmMIs/J 和 C57BI/6J. </li>
	<li> 打开气体流量到30-40%。一旦动物在大约2分钟以后明显地是无意识的, 打开气体阀门到充分的压力为另外 5 mi...

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本文介绍了一种分离完整的小鼠乳腺的技术, 用于下游组织中 ECM 表达和导管形态学的组织学分析。关于导管形态学的分析, 这一方法能够快速研究基于染色组织学切片的导管结构。导管分析的其他方法依赖于染料的注入, 使导管树的可视化, 这些方法在技术上具有挑战性和耗时。
在乳腺癌中, ECM 被报道为异常。1,2,<sup class="xr...

乳腺癌的特点是有相当程度的组织纤维化1,2,3,4。被称为 ECM, 这个泡沫塑料实体是在不同程度的发现在所有组织和主要由一个复杂的网状纤维和 non-fibrillar 胶原蛋白, 弹力素, 和糖蛋白, 除了各种信号分子, 是隔离在这个矩阵。在稳态条件下, ECM 的沉积和降解受到严格控制。5在乳腺肿瘤发生过程中, ECM 沉积和降解的平衡被破坏。因此, 据报道, 乳腺肿瘤表达了丰富的 ECM 蛋白, 如胶原、纤维连接蛋白和素等。6这些蛋白的异常表达除了增加基质交联的模式外, 还记录了促进乳腺肿瘤的进展、转移和治疗的抵抗力1、3、 4,7,8,9。
为了评估 ECM 成分和导管形态学, 对完整乳腺进行了分离。在这里, 我们使用雌性产妇小鼠缺乏 caveolin-1, 一个完整的膜蛋白已被连接到一个积极的乳腺肿瘤签名10,11,12, 并控制女性产妇 B6鼠.这些组织的组织学处理和染色允许对几种 ECM 蛋白的鉴定以及导管形态学的表征。
整体而言, 完整的乳腺隔离使研究人员有机会研究组织范围内的形态学或细胞变化, 以响应外源或内源性因素。这项技术的缺点与2维 (2D) 组织切片的分析相关联, 而不是3D 的角度, 这将使导管树的复杂形态学产生更完整的图像。鉴于乳腺内细胞细胞和细胞 ECM 相互作用的复杂性, 完整、完整的腺体分离有利于有效分析乳腺各区的导管形态学和蛋白质表达腺.

<table>
	<tbody>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus</td>
			<td>BX43</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Olympus</td>
			<td>cellSens Entry software</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5x-R Surgical Micro Loupes</td>
			<td>Rose Micro Solutions</td>
			<td></td>
			<td>Magnification at researcher&#39;s preference</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Medline</td>
			<td>DYND04035</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Rack</td>
			<td>Fisher Scientific</td>
			<td>121</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Dish</td>
			<td>Fisher Scientific</td>
			<td>112</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Fisher Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Size appropriate for tissue</td>
		</tr>
		<tr>
			<td>IHC EnVision+ Kit (HRP, Mouse, DAB+)</td>
			<td>Dako</td>
			<td>K400611-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrosirius Red Kit</td>
			<td>Abcam</td>
			<td>AB150681</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>HHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>EMD Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td>suitable for molecular biology</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1x</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>202932</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Mounting Medium</td>
			<td>Dako</td>
			<td>S1964</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#39;s Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Abcam</td>
			<td>AB23750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenascin-C antibody</td>
			<td>Abcam</td>
			<td>AB108930</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Smooth Muscle Actin antibody</td>
			<td>Abcam</td>
			<td>AB124964</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Envision Dual Link System HRP</td>
			<td>Dako</td>
			<td>K4065</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology

本程序的目的是从雌性产妇小鼠中获取 #4 腹乳腺, 以评估 ECM 表达和导管结构。在这里, 一个小的口袋下面的皮肤是创建使用梅奥剪刀, 允许分离的腺体内皮下组织的基础腹膜。3.5x 手术微放大镜的使用, 辅助了腺体的可视化。该毛皮被倒置和固定回来, 允许鉴定完整的乳房脂肪垫。每一个 #4 的腹腺都是通过滑动解剖刀刀片在皮下层和腺体之间进行了坦率的解剖。立即收获后, 腺体被放置在10% 中性缓冲福尔马林随后组织处理。切除整个腺体是有利的, 因为它主要消除了风险, 排除重要的组织范围之间的相互作用导管上皮细胞和其他微细胞的人口, 可能会错过的部分活检。该方法的缺点之一是使用固定组织的序列切片, 限制了导管形态发生和蛋白质表达对腺体内离散位置的分析。因此, 3 维 (3D) 的导管结构和蛋白质表达的变化是不容易获得的。总的来说, 该技术适用于需要完整完整的小鼠乳腺进行下游研究, 如发育性导管形态发生或乳腺癌。

雌性小鼠有5对乳腺。具体地说, 有一对宫颈腺体 (#1)、两对胸腔 (#2 和 #3)、一对腹腺 (#4) 和1对腹股沟 (#5) (图 1A)。在这里, 我们隔离了 #4 腺体, 因为它们易于辨认。在某些情况下, #4 和 #5 的腺体被孤立在一起, 因为两者之间的区别是困难的。为了隔离完整的 #4 腹乳腺, 毛皮被固定和小心地从下腹部分离出来, 揭示了由箭头指示的 #4 腺体的?...

Watch this Scientific Journal Video about Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology at JoVE.com

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

在这里, 我们提出了一个完整的, 完整的小鼠乳腺分离的协议, 以研究细胞外基质 (ECM) 的表达和导管形态学。小鼠 #4 腹腺从8-10 周大的雌性产妇小鼠中提取, 固定在中性缓冲福尔马林中, 用免疫组织化学方法对 ECM 蛋白进行切片和染色。

完整、全小鼠乳腺的分离及细胞外基质表达及腺体形态学分析

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Research

JoVE Journal

Biology

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

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

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

科研

生物学

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

变化在大鼠乳腺形态和患乳腺癌的风险

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Isolation of Mouse Salivary Gland Stem Cells

鼠标涎腺干细胞的分离

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Evaluation of Mammary Gland Development and Function in Mouse Models

乳腺的发育和功能在小鼠模型的评价

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

小鼠气管基底细胞和粘膜下腺体的导管细胞分离

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in 1. ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

导管内注射局部给药的小鼠乳腺

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

鼠心肌细胞的分离及生理分析

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

3维组织学音量和其研究小鼠乳腺腺体的应用改造

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...