我们感谢吉尔的实验室管理和技术支持, j. 霍金斯和国家卫生研究院 (NIH) 的工作人员50建立了援助老鼠护理的动物设施, 以及里德和 f. Baldrey 的行政援助。还感谢 s Motegi 和 m. Udey 分享他们的耳朵皮肤解剖协议, n. 烧伤的编辑帮助, 以及干细胞和神经血管生物学实验室成员的技术帮助和深思熟虑的讨论。KAITOKU 是日本促进科学协会 (jsp) 的支持。这项工作得到了国家心脏、肺和血液研究所 (HL005702-11 到 Y.M.) 的校内研究计划的支持。

这一节的所有实验都是在国家心脏、肺和血液研究所 (NHLBI) 动物护理和使用委员会的批准下进行的。
1. 成人小鼠耳皮收集
<ol><li>弄死成年小鼠的二氧化碳 (CO2) 暴露在一个封闭的房间, 然后确认安乐死的颈椎脱位。 注: 实验遵循国家卫生研究院 (NIH) 的安乐死方法指南。</li>
	<li>从底座解剖耳朵, 将其放置在 35 x 10 毫米的2培养皿中, 其中包含有2毫升的汉克平衡盐溶液 (HBSS)。用剪刀简单地修剪头发。</li>
	<li>从介入软骨中仔细剥离后皮肤和前皮肤。 注: 软骨附着于前皮肤。</li>
	<li>将后、前皮肤分别转移至24井板, 含1毫升冰鲜4% 多聚甲醛 (粉煤灰) 的磷酸盐缓冲盐水 (PBS) 每井。将4% 的粉煤灰中的后、前皮肤压扁。</li>
	<li>修复后和前皮肤与软骨在4°c 1 小时。</li>
	<li>在室温下, 在混合器上用温和的混合, 在1毫升的 PBS 中清洗后、前皮肤3次5分钟。</li>
	<li>将后、前皮肤与软骨转移到 35 x 10 毫米2培养皿的底部。切断基底区域, 折叠并有脂肪和结缔组织。用细弯钳剥离前皮肤软骨。</li>
	<li>用细弯镊子小心去除后皮肤内侧的毛发、脂肪组织和结缔组织。用 PBS 把皮肤弄湿。</li>
</ol>2. 小鼠耳皮肤的全层免疫组化染色
注: 以下各节中的所有实验均按照 NIH 实验室安全指南进行。
<ol><li>准备阻塞缓冲区。过滤器10% 热灭活山羊血清 (HIGS) 稀释在 pbs 与0.2% 海卫 X-100 (TX-100) 阻断缓冲, 或10% 驴血清 (DS) 稀释在 pbs 与 0.2% TX-100 阻塞缓冲区, 使用0.45 µm 过滤器单位。</li>
	<li>将后、前皮肤转移至24井板, 其中包含1毫升 10% HIGS 阻塞缓冲器或 10% DS 阻塞缓冲器。在室温下, 在搅拌机上用温和的混合孵育皮肤30分钟。</li>
	<li>在阻塞缓冲器 (10% HIGS 或 10% DS) 中稀释主要抗体 (材料表), 准备主要抗体溶液。 注: 全分队城市免疫组化分析成人耳皮肤抗体的泛神经元标记神经元特定类 III β-蛋白 (Tuj1, 兔多克隆 IgG 或小鼠 monocloncal IgG2a, 1:500 稀释在最后浓度的2µg/毫升),pan 内皮细胞标记的血小板内皮细胞黏附分子 1 (PECAM-1, 仓鼠单克隆 IgG, 1:300 稀释在最后浓度3.3 µg/毫升), 髓鞘标记髓鞘碱性蛋白 (MBP, 兔多克隆 IgG, 1:200 稀释在最后浓度的5µg/毫升) 和炎症髓细胞标记 CD11b (大鼠单克隆 IgG2b, 1:50 稀释在最后浓度20µg/毫升) 显示在图 1和图 2。以 Cy3-conjugated 抗体为血管平滑肌细胞标记α平滑肌肌动蛋白 (αSMA) 与继发抗体 (2.6) 一起孵化皮肤。我们自己测试的主要抗体在材料表中列出。来自不同物种的多种主要抗体可以同时混合。</li>
	<li>将后、前皮肤转移到含150µL 的原发抗体溶液的新井中。在4摄氏度的搅拌机上用温和的混合孵育皮肤。</li>
	<li>第二天, 将后、前皮肤转移到24井板中的新井, 或吸入含有原发抗体的阻塞缓冲液。增加1毫升的 2% HIGS 稀释在 pbs 与 0.2% TX-100 洗涤缓冲器或 2% DS 稀释在 pbs 与 0.2% TX-100 洗涤缓冲器。用3的洗涤缓冲器的变化, 每15分钟清洗皮肤, 在混合器的室温下轻轻搅拌。</li>
	<li>准备二级抗体溶液 (材料表)。在阻塞缓冲器中稀释二次抗体 (10% HIGS 或 10% DS), 并通过0.22 µm 聚偏聚氟 (PVDF) 膜注射器过滤器连接到1毫升注射器, 过滤含有二级抗体的阻塞缓冲器。 注: Alexa 488 或633共轭山羊抗兔 IgG (H + L) 或小鼠 IgG2a Tuj1, alexa 647-共轭山羊抗仓鼠 igg (h + l) 为 PECAM-1, alexa 488 共轭山羊抗兔 igg (h + l) 为 MBP, 和 Alexa 594 共轭大鼠 igg (h + l) 为 CD11b 使用以1:250 稀释在最后的集中8µg/毫升。皮肤用 Cy3-conjugated αSMA 抗体 (1:500 稀释在最后浓度的2-3 µg/毫升) 与这些继发抗体结合而孵化。</li>
	<li>离心溶液在 1.3万 x g 10 分钟内从阻塞缓冲器中去除二次抗体的聚集粒子。 注: 不同种类的荧光共轭二次抗体可同时混合。</li>
	<li>将后、前皮肤转移至含150µL 的二级抗体溶液。在铝箔上包装24井板, 以避免光和孵化的皮肤为 1 h 与温和的混合在一个搅拌机在室温下。</li>
	<li>将后、前皮肤转移到24井板中的新井, 或完全吸管二次抗体溶液。添加1毫升的 2% HIGS 洗涤缓冲器或 2% DS 洗涤缓冲器。</li>
	<li>在铝箔上包裹24井板, 用洗涤缓冲器的3变化每15分钟清洗一次, 在室温下混合搅拌。</li>
</ol>3. 在幻灯片上安装耳皮
<ol><li>将皮肤放在 35 x 10 毫米2培养皿的底部。用低光照显微镜下的细弯钳, 从皮肤内侧小心去除毛发、脂肪组织、结缔组织、灰尘和纤维, 以尽量减少广泛的照片漂白。用 PBS 把皮肤弄湿。</li>
	<li>使用镊子将皮肤转移到粘附显微镜滑动。将后部和前部皮肤与内侧面朝上滑动。用镊子将皮肤平整。</li>
	<li>在液体防褪色安装介质中安装皮肤, 以避免漂白和保存荧光信号。确保没有气泡在皮肤上或周围。</li>
	<li>在皮肤标本上仔细盖上盖玻片, 在室温下将装入的皮样幻灯片放在黑暗中, 让安装介质变得牢固。用指甲油将盖玻片密封到滑轨上, 并将其存放在4摄氏度, 用于长期贮存。</li>
</ol>4. 共焦显微术
<ol><li>为显影设置合适的激光器。本实验采用三激光源 (氩 488 nm、DPSS 561 nm 和 HeNe 633 nm) 共焦显微镜。</li>
	<li>使用顺序扫描工具, 同时激发三重染色样品, 以避免和减少任何重叠。 注意: 图像将使用顺序扫描模式以顺序方式进行。</li>
	<li>10X 目标下的图像。使用平铺扫描工具捕捉整个耳朵皮肤。设置 z 堆栈, 并确保 z 位置覆盖整个耳朵皮肤的厚度。</li>
</ol>

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作者没有什么可透露的。

本协议描述成人耳皮肤的全芒 immunonohistochemical 成像, 用于分析神经血管结构和免疫细胞分布。我们认为, 这种方法有许多实验优势, 研究人员的分支形态发生和模式的周围神经和血管, 以及三维分布的皮肤成分, 包括免疫细胞和头发毛囊.图像的结果可以用成像软件进行量化, 进一步定量分析。
正确地制备耳部皮肤对本议定书的成功至关重要。首先, euthanizing 后应仔细解剖耳朵皮...

皮肤由三层组成: 表皮、真皮和皮下组织。它已被作为一个模型系统来研究干细胞的维持, 分化和形态发生的发展, 以及再生, 肿瘤发生和炎症的成人。皮肤有丰富的血管化和支配, 使周围神经系统和血管系统的发育协调良好。
我们以前已经展示了一个完整的胚胎皮肤成像技术与多重标记研究完整的周围神经和血管, 包括其蜂窝组件1,2,3,4: 血管中的感觉轴突、交感神经轴突、神经的雪旺细胞、内皮细胞、毛细血管和血管平滑肌细胞 (VSMCs)。在血管生成过程中, 一个主要的毛细管网络经过密集的血管重塑, 发展成为一种分层的血管分支网络。在发育的真皮/皮下组织, 动脉分支与周围的感觉神经和静脉形成相邻的动脉。在分层的血管网络被 VSMCs 彻底覆盖后, 交感神经延伸沿和支配大口径血管1,5,6。尽管发展中的神经和血管系统之间的密切联系具有重要意义, 但一个主要问题是解决成人不同病理情况下神经血管网络的发生情况。三维高分辨率成像是必要的, 以了解发病机制, 以及解剖可识别的分支形态发生和模式。
组织切片染色法分析成年小鼠皮肤的神经细胞和血管形态。其他研究还使用了整个影像的皮肤来可视化周围神经和血管, 除了毛囊, 皮脂腺, 和 arrector 毛肌肉7,8,9。然而, 成人皮肤的厚度使得它很难分析整个深度的皮肤。
在本研究中, 我们开发了一种新的高分辨率的成人耳皮肤的全安装成像, 以克服这些挑战。耳朵皮肤是容易接近的解剖和后续整个安装成像的皮肤在其整个深度。因此, 它是一个简单的和高度重复的方法, 可用于比较三维结构的周围神经和血管系统的皮肤, 与综合量化测量。结果表明, 成人皮肤中保留了大口径血管对周围感觉和交感神经的定位。该协议的目标是可视化分支形态发生, 以及周围神经和血管的模式, 以及在各种情况下, 如炎症和成年小鼠模型的细胞水平上的免疫细胞分布。再生.

<table>
	<tbody>
		<tr>
			<td>10 x Phosphate Buffered Saline</td>
			<td>KD Medical</td>
			<td>RGE-3210</td>
			<td>PBS, without Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>HBSS, with Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>PFA, fixative, diluted in PBS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Gibco</td>
			<td>16210064</td>
			<td>Component of blocking/washing buffer</td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Immuno research</td>
			<td>017-000-121</td>
			<td>Component of blocking/washing buffer</td>
		</tr>
		<tr>
			<td>Curved fine tweezers</td>
			<td>Dumont</td>
			<td>RS-5047</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved tweezers</td>
			<td>Integra Miltex Vantage</td>
			<td>V918-782, V918-784</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter Unit 0.45 mm</td>
			<td>Thermo Scientific</td>
			<td>157-0045</td>
			<td>For filtration</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Coviden</td>
			<td>8881501400</td>
			<td>For filtration</td>
		</tr>
		<tr>
			<td>Syringe filter Unit 0.22 mm</td>
			<td>Millex-GV</td>
			<td>SLGVR04NL</td>
			<td>For filtration</td>
		</tr>
		<tr>
			<td>ProLong Gold</td>
			<td>Thermo Scientific</td>
			<td>P36934</td>
			<td>Anti-fade mounting medium</td>
		</tr>
		<tr>
			<td>Nail Polish</td>
			<td>Electron Microscopy Sciences</td>
			<td>72180</td>
			<td>For sealing</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>MZ95</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>TCS SP5</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop CC 2017</td>
			<td>Adobe</td>
			<td></td>
			<td>Graphics editor software</td>
		</tr>
		<tr>
			<td>Illustrator CC 2017</td>
			<td>Adobe</td>
			<td></td>
			<td>Graphics editor software</td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>NIH</td>
			<td></td>
			<td>Image processing software</td>
		</tr>
		<tr>
			<td>Anti-PECAM-1 (CD31) antibody</td>
			<td>Millipore</td>
			<td>MAB1398Z</td>
			<td>Hamster IgG, vascular endothelial cell marker, 1:300</td>
		</tr>
		<tr>
			<td>Anti-PECAM-1 (CD31) antibody</td>
			<td>BD Pharmingen</td>
			<td>553369</td>
			<td>Rat IgG2a kappa, vascular endothelial cell marker, 1:300</td>
		</tr>
		<tr>
			<td>Anti-aSMA antibody conjugated with cy-3</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Mouse IgG2a, vascular smooth muscle cell marker, 1:500</td>
		</tr>
		<tr>
			<td>Anti-EphB1 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-9319</td>
			<td>Goat polyclonal, venous endothelial cell marker, 1:100</td>
		</tr>
		<tr>
			<td>Anti-neuron-specific Class III b-tubulin (Tuj1)</td>
			<td>Abcam</td>
			<td>AB18207</td>
			<td>Tuj1, Rabbit polyclonal IgG, pan-axonal marker, 1:500</td>
		</tr>
		<tr>
			<td>Anti-Tuj1 antibody</td>
			<td>Covance</td>
			<td>MMS-435P</td>
			<td>Mouse IgG2a, pan-axonal marker, 1:500</td>
		</tr>
		<tr>
			<td>Anti-MBP antibody</td>
			<td>Abcam</td>
			<td>AB40390</td>
			<td>Rabbit polyclonal IgG, myelination marker, 1:200</td>
		</tr>
		<tr>
			<td>Anti-Tyrosine Hydroxylase antibody</td>
			<td>Chemicon</td>
			<td>AB152</td>
			<td>Rabbit polyclonal, sympathetic neuron marker, 1:500</td>
		</tr>
		<tr>
			<td>Anti-Peripherin antibody</td>
			<td>Chemicon</td>
			<td>AB1530</td>
			<td>Rabbit polyclonal, peripheral neuron marker, 1:1000</td>
		</tr>
		<tr>
			<td>Anti-CD11b antibody</td>
			<td>Bio-Rad</td>
			<td>MCA74G</td>
			<td>Rat IgG2b, inflammatory cell marker (macrophages), 1:50</td>
		</tr>
		<tr>
			<td>Anti-CD45 antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-0451-85</td>
			<td>Rat IgG2b kappa, pan-hematopoietic cell marker, 1:500</td>
		</tr>
		<tr>
			<td>Anti-CD3 antibody</td>
			<td>Bio-Rad</td>
			<td>MCA1477T</td>
			<td>Rat IgG1, immune cell marker, 1:100</td>
		</tr>
		<tr>
			<td>Anti-CD45R (B220) antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-0452</td>
			<td>Rat IgG2a kappa, inflammatory cell marker, 1:200</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11122</td>
			<td>Rabbit polyclonal, 1:300</td>
		</tr>
		<tr>
			<td>Anti-GFP antibody</td>
			<td>Abcam</td>
			<td>Ab13970</td>
			<td>Chicken polyclonal, 1:500</td>
		</tr>
		<tr>
			<td>Anti-b-gal antibody</td>
			<td>Cappel</td>
			<td>55976</td>
			<td>Rabbit polyclonal, 1:5000</td>
		</tr>
		<tr>
			<td>Anti-RFP antibody</td>
			<td>Abcam</td>
			<td>Ab62341</td>
			<td>Rabbit polyclonal, 1:300</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG (H+L) Alexa 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11034</td>
			<td>Rabbit polyclonal secondary antibody, 1:250</td>
		</tr>
		<tr>
			<td>Goat anti-hamster IgG (H+L) Alexa 647</td>
			<td>Jackson Immuno research</td>
			<td>127-605-160</td>
			<td>Hamster polyclonal secondary antibody, 1:250</td>
		</tr>
		<tr>
			<td>Goat anti-rat IgG (H+L) Alexa 594</td>
			<td>Jackson Immuno research</td>
			<td>112-585-167</td>
			<td>Rat polyclonal secondary antibody, 1:250</td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG2a Alexa 633</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21136</td>
			<td>Mouse IgG2a secondary antibody, 1:250</td>
		</tr>
	</tbody>
</table>

whole-mount confocal microscopy for adult ear skin: a model system to study neuro-vascular branching morphogenesis and immune cell distribution

在这里, 我们提出了一个完整的成人耳皮肤成像技术的协议, 以研究全面的三维神经血管分支形态发生和模式, 以及免疫细胞分布的细胞水平。对成人组织周围神经和血管解剖结构的分析, 为了解创伤愈合等病理条件下的功能性神经血管接线和神经血管变性提供了一些见解。作为一个高度信息的模型系统, 我们把我们的研究集中在成人耳皮肤, 这是容易被解剖。我们的简单和可重现的协议提供了一个准确的描述整个皮肤的细胞成分, 如周围神经 (感觉轴突, 交感神经轴突, 和雪旺细胞), 血管 (内皮细胞和血管平滑肌细胞) 和炎症细胞。我们相信这项议定书将为调查不同病理条件下成人耳皮肤的形态学异常和周围神经和血管的炎症铺平道路。

成年小鼠后耳皮肤 (图 1A) 和前耳皮肤 (图 1B) 与αSMA (红色)、Tuj1 (绿色) 和 PECAM-1 (蓝色) 的抗体 immunostained。后皮肤 immunostained 研究神经免疫分布, 使用抗体 CD11b (红色) 和 MBP (绿色), 连同 Tuj1 (蓝色) (图 2A)。在单个细胞分辨率 (图 2B) 中检测到 CD11b+炎症细胞 (包括巨噬细胞) 的分?...

Watch this Scientific Journal Video about Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution at JoVE.com

Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

在这里, 我们描述了一个高分辨率全安装成像方法在整个成年小鼠耳皮肤, 这使我们能够想象分支形态发生和模式的周围神经和血管, 以及免疫细胞分布。

成人耳皮肤全安装共焦显微术: 一种研究神经血管分支形态发生和免疫细胞分布的模型系统

Here, we present a protocol of a whole-mount adult ear skin imaging technique to study comprehensive three-dimensional neuro-vascular branching ...

whole-mount-confocal-microscopy-for-adult-ear-skin-model-system-to

Research

JoVE Journal

Developmental Biology

13.0K 次观看.  National Institutes of Health. 在这里, 我们描述了一个高分辨率全安装成像方法在整个成年小鼠耳皮肤, 这使我们能够想象分支形态发生和模式的周围神经和血管, 以及免疫细胞分布。

视频: Whole-mount Confocal Microscopy for Adult Ear Skin: A Model System to Study Neuro-vascular Branching Morphogenesis and Immune Cell Distribution

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

一种简便有效的成人皮肤组织中人角质形成细胞的分离方法

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...