我们要感谢协助试剂制备丽贝卡Paquette。 MPM是一个露丝L. Kirchstein predoctoral奖学金的赠款GM - 41514（MDC），NS - 48252（KGC），GM - 48071也从美国国立卫生研究院（IP）的健康和支持国家机构的支持。

1。细胞过继转移： 尾静脉或眼内注射引入细胞的跟踪标记或荧光蛋白表达的淋巴细胞都合适的方法。在这个视频。我们展示了尾静脉注射的细胞过继转移。 A.材料 CD4 + T细胞与1.4μm的30分钟37 ° C CFSE标记，洗涤两次，重悬在100μL的RPMI - 1640。细胞被装入一个无菌的28针注射胰岛素的注射器。正在做实验使用的细胞数量取决于。通常情况下，5万T细胞是够简单的T细胞的可视化。需要一个鼠标灭鼠限位，以防止受伤的动物和自己。 B.保护动物鼠标是继转移担保慢慢备份到啮齿类动物限位的动物（见视频）和关闭的钢管的开口端，轻轻按下柱塞。不要让去的老鼠尾巴，在此过程中，一些较小的动物可以打开里面的限位。这种方法可以防止伤害自己和在注射的动物。成立灭鼠限位时，不推进一步柱塞比是要不断向前跳跃的动物。此外，不推柱塞对动物，和不留在限位长时间的动物。 C.尾静脉可视化轻轻拉出，以便它轻轻地包裹在你的食指和你的拇指对手指内举行对你的尾巴。确定位于直立的尾巴两侧上的尾静脉。可视化是促进变暖尾用温水，然后用乙醇擦拭。 D.注射细胞当已经找到静脉，插入注射器的锥端，在一个浅的角度平行静脉。一旦在静脉，轻轻压下柱塞，如果​​你在静脉应注射无阻力。如果有任何抵抗，取下注射器，并在不同的位置后再试一次。 如果你在静脉，慢慢地压低活塞，直到注射完成。尾巴不应该成为在注射肿胀。如果确实如此，这将意味着，在静脉针。这是可能的“输”在静脉注射。如果发生这种情况，去掉针头，并尝试在不同部位注射。如果你需要尝试超过一次注射，最好是迈向鼠标身体的尾巴从原来的注射部位。这种可能性的计划提前启动远端的尾巴上给自己另一个企图在继转移的房间。 E.注射后注意事项注射完毕后，将出现一个小的静脉血液的回流，从。轻轻按下注射部位，用干净的擦，直到出血停止。取出柱塞，让动物向前走，走出了限位，一边轻轻控股尾巴上。 请务必检查您的动物保健委员会和动物使用的协议，以确保您遵守。重要的是实践这项技术，真实的实验，然后再尝试几次。 2。淋巴结隔离两个光子成像淋巴结分离需要小心正确的淋巴结周围组织切除。 A.选择和周围淋巴结的位置成像淋巴结的选择将取决于实验的目标。局部感染或注射淋巴结，并选择适当的节点成像。由Van登布洛克等文章，介绍了3详细的小鼠淋巴结的位置和形态。在这段视频中，我们展示了六个大型的外周淋巴结，这是适合于细胞的分离或成像切除。 B.淋巴结清除： 记住组织的完整性，同时消除淋巴结。要小心，不要胶囊破裂或以任何方式损害淋巴结结构。淋巴结的结构完整性的丧失会危及实验。如有必要，删除解剖（＃5杜蒙）在解剖显微镜下用细钳从淋巴结表面脂肪。淋巴结不应该有任何节点表面上剩余的脂肪，因为这会掩盖散射光成像。 适当切除淋巴结也是一个重要的技术，实践前的第一个真正的实验。 3。淋巴结的Nod发送图像的设置和注意事项应保证成像阶段，在这种情况下由一个凹腔淋巴结。温暖，氧气灌注媒体是从一个侧面，用蠕动泵室和内联加热器，泵入和退出真空管废物收集烧瓶通过向下分级进入收集室通道。在我们的系统，淋巴结被淹没在37 ° C媒体和医疗级carbogen（5％，95％的CO 2，O 2）超灌注。灌注介质的温度应尽可能接近淋巴结记录。您的特定系统的，可能需要在媒体流量和舞台设计的变化，以适应显微镜阶段和客观。 A.淋巴结成像设置： 在视频所示，我们安全第一附加的淋巴结，髓质的一面朝下（图像T或B细胞区使用一个直立显微镜），切成适当大小的塑料盖玻片。重要的是要使用无毒的组织胶（我们使用3M公司VetBond™）。 在流动媒体是安全的盖玻片盖玻片底面放置在一个小民建联硅脂，然后坚持这种成像室的玻璃基板。 B.双光子成像的考虑： 有几个因素可能会导致细胞停止移动：温度过高或过低的激光功率过剩，压缩或者其他损害的淋巴结;和缺乏氧气。由于激光功率过高或温度所造成的损坏&gt;〜39 ° C是不可逆转的。 如果细胞是暗淡的，它通常是更好的增加探测器的增益，而不是使用一个更高的激光功率可视化细胞的细胞标记协议。优化标签或荧光蛋白的表达，可能需要把上述背景自发荧光的水平的荧光。有了合理的增益和激光功率设置日志基线以上的荧光转变，流式细胞仪检测细胞可视化的合理。 可调谐双光子激光器，激发波长应少一点一倍以上的单光子激发荧光成像的最大。在使用一个以上的标签或荧光蛋白的双光子实验，它可能是要用来寻找最佳设置两个标签的激发波长的实验。双光子激发，也可以用于图像的内源性胶原蛋白结构的内在蓝色的二次谐波产生的优势。二次谐波（SHG）发生在两个光子在材料相结合，并发出两倍于原来的光子的频率的单光子。在组织中，发生双光子激发时，入射激光光线垂直于一个高度有序的蛋白质结构，如胶原蛋白。 在预览评论4，5，关于双光子成像的实际问题进行了讨论。

<ol>
	<li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+Cell+Motility+and+Interaction+Dynamics+Imaged+by+Two-Photon+Microscopy+in+Lymphoid+Organs.+Annu+Rev+Immunol.">Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs. Annu Rev Immunol.</a> Annu Rev Immunol. , (2008).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Van den Broeck, W., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> J Immunol Methods. 312, 12-19 (2006).</li><li>Cahalan, M. D., Parker, I., Wei, S. H., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+tissue+imaging:+seeing+the+immune+system+in+a+fresh+light.">Two-photon tissue imaging: seeing the immune system in a fresh light.</a> Nat Rev Immunol. 2, 872-880 (2002).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li></ol>

在这个视频协议，我们将展示收养细胞转移和淋巴结成像在周围淋巴结淋巴细胞活力所需的隔离和准备程序。双光子成像有几个方面的共焦成像的优势，都explanted组织（如图所示），并在活体准备。值得注意的是，使用红外线激发，减少了光散射和淋巴结的囊下的成像〜300微米，允许在一些组织4更深。此外，多光子激发被限制的客观的焦点，最大限度地减少照片的褪色和组织损伤出方焦平面。然而，正如任何新技术，双光子成像是不是万能的，它的局限性和潜在的隐患，必须保持头脑5。其中之一是，要求 - 除了利益的胶原蛋白细胞，如二次谐波产生的内在信号必须由外在探头荧光标记或荧光蛋白的表达。因此，给人的印象是这些标记的细胞在一个黑色的虚空游泳;而事实上，他们都沉浸在和一个复杂的环境中的结构元素和无数其他的未标记的，无形的细胞交互。 在双光子成像介绍免疫以来的六年中，这种技术允许研究人员直接在可视化的完整的活组织的过程，包括细胞间的相互作用，细胞运动和本地化，细胞信号通路和细胞毒性细胞，以解决长期存在的问题杀害事件。领域已经超出最初的现象学描述的迅速发展，并与计算机建模和仿真定量分析，现在开始做出显然是混乱的蜂窝议案和淋巴结内的相互作用如何导致一个有效的免疫反应的的感觉。这种进步是全面检讨在最近出版的1，它列出了100多个描述双光子显微镜的immunoimaging应用程序文件。

dissection and 2-photon imaging of peripheral lymph nodes in mice

双光子成像揭示了一个优雅的芭蕾舞蹈艺术的活力和细胞相互作用，在基础条件下的淋巴结，并启动免疫反应1。在这里，我们目前继转移标记的T细胞，淋巴结肿大隔离，成像蠕动的CD4 + T细胞在explanted淋巴结作为第一个在2002年2的方法。双光子成像需要的免疫细胞，细胞荧光标记，与细胞跟踪染料染色，或表达一种荧光蛋白。我们展示的注射细胞过继转移的过程，从供体小鼠的尾静脉进入收件人的动物，在那里，他们家约15-30分钟内淋巴器官。我们说明了一个淋巴结的隔离和描述方法，以确保正确安装切除淋巴结。其他考虑因素，如适当的氧合灌注介质，温度，激光功率进行了讨论。最后，我们目前的3D视频影像的幼稚CD4 + T细胞参展稳态活力在37 ° C。

Watch this Scientific Journal Video about Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice at JoVE.com

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

双光子成像内已发现的基础条件下的淋巴结和淋巴细胞的活力和细胞相互作用过程中的免疫反应1。在这里，我们证明收养的T细胞，淋巴结的隔离和成像蠕动的CD4 + T细胞在explanted淋巴结转移。

夹层和小鼠的外周淋巴结的双光子成像

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

dissection-and-2-photon-imaging-of-peripheral-lymph-nodes-in-mice

Nanyang Technological University

Research

JoVE Journal

Biology

46.1K 次观看.  University of California, Irvine (UCI). 双光子成像内已发现的基础条件下的淋巴结和淋巴细胞的活力和细胞相互作用过程中的免疫反应1。在这里，我们证明收养的T细胞，淋巴结的隔离和成像蠕动的CD4 + T细胞在explanted淋巴结转移。 

视频: Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

分离的CD4 +从小鼠淋巴节点使用美天旎MACS纯化的T细胞

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

科研

生物学

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

在胸腺的鼠标采用双光子显微镜的原位成像

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

通过颅窗口在新皮层的血流量为双光子成像方法

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

代骨髓衍生的双光子成像使用的小鼠树突状细胞

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

功能成像在小鼠体内的棕色脂肪与FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖，成像和药物治疗: 果蝇蛹睾丸和单身男性生殖系囊肿的体外培养

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

在出生后的小鼠听泡解剖:中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...