Name: preparation of murine submandibular salivary gland for upright intravital microscopy
Uploaded: 2018-05-07T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

这项工作由瑞士国家基金会 (SNF) 项目赠款31003A_135649、31003A_153457 和 31003A_172994 (JVS) 提供资金, Leopoldina 2011-16 (至 BS)。这项工作得益于在伯尔尼大学的 "显微成像中心" (MIC) 的光学设置。

所有动物的工作都已得到州动物试验委员会的批准, 并根据联邦准则进行。
1. 麻醉鼠标
<ol><li>佩戴个人防护用品, 包括实验室外套和手套。</li>
	<li>将氯胺酮、甲苯噻嗪和生理盐水混合在一起, 其工作浓度分别为20毫克/毫升和1毫克/毫升。将工作库存腹腔 (ip) 注入8-10 µL/克鼠标。把鼠标放回笼子里。 注: 本协议已测试6至40周的男性和女性小鼠的 C57BL/6 背景。</li>
	<li>准备乙酰...

<ol>
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该协议提供了一个简单的方法,在体内成像的小鼠颌下腺和舌下涎腺使用直立非线性显微镜经常使用的免疫学领域。该方法可用于头颈区其他外分泌腺体的成像。例如, 我们的实验室以类似的方式对泪腺进行了成像 (不显示)。
除去在 SMG 周围的结缔组织是这个协议最关键的步骤, 因为意外的组织损伤可能发生。由于 smg 的组织会变得缺氧, 因此对 smg 的主要血液供应造成的?...

唾液由外分泌腺体分泌来润滑食物, 保护口腔粘膜表面, 并提供消化酶和抗菌物质1,2。除了在口腔黏膜下层散置的小涎腺外, 根据其位置1、2, 还有三个双侧的主要腺体被确定为腮腺、舌下和颌下腺。金字塔状上皮细胞, 组织成烧瓶状囊 (腺泡细胞) 或 demilunes 包围的上皮细胞和基底膜, 分泌的浆液和粘液成分的唾液1。腺泡细胞狭窄的腔内空间排水成夹层导管, 直到它们最终加入一个排泄管道1。SMG 的主要排泄管称为沃顿商学院的管道 (WD), 并打开到舌下阜3,4。因此, SMG 上皮隔间是一个高度 arborized 的结构, 具有流形终端端点, 类似于一束葡萄1,5,6。SMG 间质由血液和淋巴管组成, 内含于结缔组织7中, 内含副交感神经8和细胞外基质 5. 正常的人和啮齿动物的涎腺也含有 T 细胞, 巨噬细胞和树突状干细胞9, 以及分泌免疫球蛋白 A (IgA) 到唾液9,10的血浆细胞。由于其在健康和疾病方面的多方面作用, SMG 在许多生物研究领域都有兴趣, 包括牙科4、免疫学11、肿瘤学12、生理学8和细胞生物学3。
动态细胞过程和交互的成像是生物学研究中的一个强有力的工具13,14。基于非线性光学 (NLO) 的深部组织成像和创新 inmicroscopes 的发展, 它依赖于通过样本对多个光子的散射或吸收, 从而可以直接检查复杂组织中的细胞过程13 ,15。多光子的吸收涉及用低能光子传递总励磁能量, 这限制了荧光对焦平面的激发, 从而允许更深层的组织穿透, 减少了光损伤和噪声。励磁13,15。这一原则采用双光子显微镜 (2PM), 并允许在高达1毫米15,16深度的荧光标本上进行成像。虽然商用2PM 设置已变得方便和可靠, 活体成像的主要挑战是仔细暴露和稳定麻醉小鼠的靶器官, 特别是对时间失效序列的成像。数据获取后数字漂移校正的几种方法已发布17,18和我们最近开发了 "VivoFollow", 一个自动校正系统, 它抵消缓慢的组织漂移实时使用计算机化阶段19。然而, 对于高质量的成像, 要尽量减少组织运动, 尤其是呼吸或心跳导致的快速运动, 这仍然是至关重要的19。为多个器官 (包括脊髓20、肝脏21、皮肤22、肺23和淋巴结24) 发布了准备和稳定程序。此外, 大鼠涎腺成像模型已经开发了3,25和进一步完善的高分辨率活体成像的小鼠 SMG 量身定做的倒置显微镜设置26,27,28。
在这里, 我们提出了一个实用的和适应性的协议, 活体成像的小鼠的直立非线性显微镜, 这是普遍用于活体成像在免疫学领域。为此, 我们改良了一个广泛使用的固定化阶段用于腘淋巴结准备。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Narketan 10 %&#160; (Ketamine) 20ml (100 mg/ml)</td>
			<td>Vetoquinol</td>
			<td>3605877535982</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine) 25 ml (20 mg/ml)</td>
			<td>Bayer</td>
			<td>680538150144</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline NaCl 0.9%</td>
			<td>B. Braun</td>
			<td>3535789</td>
			<td></td>
		</tr>
		<tr>
			<td>Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)</td>
			<td>Fatro</td>
			<td>6805671900029</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Wahl</td>
			<td>9818L</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Veet</td>
			<td>4002448090656</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue Ultra gel, instantaneous glue</td>
			<td>Pattex, Henkel</td>
			<td>4015000415040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass slides 20 mm and 22 mm</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>631-1343/ 631-1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Grease for laboratories 60 g glisseal N</td>
			<td>Borer (VWR supplier)</td>
			<td>DECO514215.00-CA15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>14090-09</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>11252-20</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Migros</td>
			<td>617027988254</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Gauze Gazin 5 x 5 cm</td>
			<td>Lohmann and Rauscher</td>
			<td>18500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Texas Red dextran 70kDa</td>
			<td>&#160;Molecular Probes</td>
			<td>D1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade Blue dextran 10kDa</td>
			<td>invitrogen</td>
			<td>D1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon system</td>
			<td>LaVision Biotec</td>
			<td>TrimScope I and II</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>XLUMPLANFL 20x/0.95 W objective</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td>or other water immersion objective&#160;</td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Fluke</td>
			<td>95969077651</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of murine submandibular salivary gland for upright intravital microscopy

颌下腺 (SMG) 是三大涎腺之一, 对许多不同领域的生物研究, 包括细胞生物学, 肿瘤学, 牙科和免疫学感兴趣。SMG 是由分泌上皮细胞、肌、内皮细胞、神经和细胞外基质组成的分泌腺。大鼠和小鼠的动态细胞过程以前已经被成像, 主要是使用倒置的多光子显微镜系统。在这里, 我们描述了一个简单的协议, 以手术准备和稳定麻醉小鼠在体内的在活体成像与直立多光子显微镜系统。我们提出了具有代表性的活体图像集内源性和过继转输转移荧光细胞, 包括标签的血管或唾液导管和第二次谐波生成, 以可视化纤维胶原。总之, 我们的协议允许手术制备小鼠涎腺在直立显微镜系统, 这是通常用于活体成像在免疫学领域。

该协议允许成像几乎所有的背部或腹侧的 SMG。视野通常也包括舌下涎腺, 这与在细胞成分中的 SMG 略有不同4。两个腺体由纤维胶原蛋白封装, 并细分成裂片。大多数2PM 系统可以通过测量 2nd谐波信号来产生纤维胶原的无标签图像, 但通常需要荧光分子来可视化细胞和亚型结构。例如, 绿色荧光蛋白 (GFP) 的无处不在的转基因表达与 2nd谐波信?...

Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

直立活体显微术制备小鼠颌下腺涎腺

我们描述了一个协议, 以手术揭露和稳定的小鼠颌下腺腺活体成像使用直立活体显微镜。该协议很容易适应小鼠和其他小啮齿目动物头部和颈部区域外分泌腺体。

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

腹股沟淋巴结的活体显微镜

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

科研

免疫与感染

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

活体显微镜的脾：寄生虫的流动性和血流量的定量分析

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

生发中心的高度已解决活体条纹照明显微镜

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

肝脏活体如下显微成像<em&gt;利什曼原虫</em&gt;感染：肝脏血流动力学的评估

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

免疫细胞健康和疾病肝脏使用CXCR6.Gfp记者小鼠长期活体多光子显微镜成像

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

A Method for the Measurement of Salivary Gland Function in Mice

小鼠涎腺功能的测定方法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...