Name: microdissection of black widow spider silk-producing glands
Uploaded: 2011-01-11T23:59:00.000+00:00
Duration: 9 min 47 s
Description: Watch this Scientific Journal Video about Microdissection of Black Widow Spider Silk-producing Glands at JoVE.com

这项工作是由一个NSF的锐格兰特MCB - 0950372题为黑寡妇蜘蛛丝的分子表征支持。

1。麻醉蜘蛛腹部和隔离<ol><li>蜘蛛从一个玻璃瓶转移到一个纸箱内衬塑料（图1A）。我们通常从woodpiles，车库或灌木丛中收集的蜘蛛和玻璃罐的实验室，他们的房子。由于蜘蛛不能爬上塑料衬里，它提供了一个有效的方法来从一个玻璃瓶或咖啡可以转移到一个较小的小瓶anesthetizing目的的蜘蛛。虽然处理的蜘蛛，在这一点上，你应该穿两双乳胶手套或为安全起见，园艺手套。 </li><li>虽然蜘蛛衬塑框，方法从背面的蜘蛛，让蜘蛛幻灯片的塑料培养瓶。蜘蛛后进入小瓶，立即将插头在上面（图1B）。我们建议使用一小瓶，大约为101.6毫米x 31.75毫米（长x宽）。这最大限度地减少二氧化碳气体的anesthetizing步骤所需的金额。 </li><li>麻醉的蜘蛛，在每平方英寸5-10磅二氧化碳气体为10分钟（图1C）。这种气体的数量只会敲蜘蛛5分钟左右，因此麻醉后，你必须立即进行步骤1.4-1.5。 </li><li>放置到一个小夹层菜的麻醉蜘蛛和使用产钳的位置蜘蛛背的一面向上（红色沙漏朝下）。 </li><li>夹蒂（窄柄连接头胸部和腹部）释放其余的蜘蛛（图1D）的腹部。在培养皿过夜存放在冷冻的头胸部和丢弃后冻结的第二天。要小心蜘蛛的毒牙是目前这一细分市场上移动时头胸部。 </li><li>拧紧腹部小夹层菜Sylgard材料，使用单一的昆虫针。通过从花梗裁剪（图1E）的孔插入昆虫针。这可以很容易地被认定为流体的地点是从剪裁的花梗新兴。确保的喷丝板（花梗的另一端）正朝着面向底部离您最近的。 </li></ol> 2。拆除外骨骼<ol><li>通过使用的microscissors对外骨骼的第一个切口，在孔从花梗裁剪开始。横向切割，直到背侧双方达成（图1F）。使用一对镊子，剥回前的外骨骼段，并在这个位置插入第二个接脚。第二针插入，打开从最初的横向切割解剖盘尾端（90 °角），并继续切割，直到达成的喷丝板（图1G）。这个切口将垂直或垂直的初始横向切割。切不要通过纵切口时，喷丝板（后相对的沙漏，它有一个圆形的外观）。如果不插入第二针，将导致在纺纱时垂直切口进行腹部。 </li><li>淹没腹部解剖缓冲液（0.1 M的氯化钠，柠檬酸钠0.015中号，0.1％焦碳酸二乙酯）。 </li><li>剥离剩余的外骨骼开始从最初的，外侧切口。当这是完成后，脂肪组织应清晰可见（图1H）。 </li></ol> 3。丝绸生产腺体隔离<ol><li>使用镊子，开始戏弄了脂肪层（图1H）。脂肪将有一个淡黄色的外观发白​​。继续，直到大部分的脂肪和丝腺都清晰可见。 tubuliform腺体组成的三双很长，圆柱形管，腹腔丰富。主要ampullate，这是对发现的，是一个大的，红新月会令人费解的长尾巴和远端近端管，宽度减小，因为它接近蜘蛛的喷丝板形壶腹。轻微ampullate是非常相似的形态主要ampullate的，但它明显变小。 flagelliform腺，也发生在对圆形和一个小圆柱锯齿形管走向的喷丝板向下延伸multilobular。总腺（对）是一个非常大的，multilobular的腺体，有一个非常大的的排泄管上有大的，不规则的裂片。 aciniform腺体发生在大量类似于短，小的指状的预测。梨状腺是最小的腺体在腹部，并有许多板结，类似于一个有许多小的排泄管道，向下延伸的喷丝板的风扇的圆柱形管道。 </li><li>我们建议删除的腺体按照以下顺序：tubuliform，主要ampullate，flagelliform，轻微的ampullate，骨料，aciniform，然后梨状（图2A - B的）。 </li><li>开始与tubuliform挑逗这些腺体休息日Ë腺体 - 他们应该坐在其他腺体的顶部（图2A）。在一个单一的蜘蛛有三种tubuliform腺体对。允许他们自由浮动的自由，但仍其管道连接到他们的喷丝板。 </li><li>下一步，梳理了主要ampullate腺体。主要有两个ampullate腺体之一蜘蛛。在搬迁过程中，确保保持连接导管的腺体（图2A）。 </li><li>继续梳理其余的腺体，远离对方，工作仔细，以避免穿刺腺体，尤其是汇总和flagelliform腺体。每个腺应该是自由浮动的喷丝板的外骨骼中的所有，它的退出点。还发现在对这些腺体。取出捏钳管道，并仔细拉，直到它打破了从出口点，每个腺。在对主要ampullate，flagelliform，未成年人ampullate，总腺体。 aciniform和梨状腺体发生多对。 </li><li>取出捏钳管道，并仔细拉，直到它打破了由喷丝头向下从出口点，每个腺。我们通常存储腺体湿​​的，但是，我们不添加额外的缓冲区。 </li><li>由于腺体中取出，放入无菌预先标记的1.5毫升离心管。个别腺体显示在图3A - G。保持这些腺体在冰上，然后闪在液氮冻结。以下闪光灯在液氮冷冻，放置在-80 ° C的存储。 </li></ol> 4。代表性的成果消除不同的腺体时，应格外小心，同时处理flagelliform和总腺体，因为这两种结构可以很容易刺破和损坏的镊子。此外，这是值得注意的形态flagelliform和汇总的腺体看起来非常相似，他们搬迁前，他们往往是相互交织在一起。为了防止在去除这些组织的交叉污染，应仔细挑逗剥离这些结构分开。此外，初次接触后的丝绸生产腺体，aciniform和梨状腺体将是最困难的立即可视化由于其较小的尺寸和解剖位置。经常有许多鸡蛋的存在。此外，应采取额外的护理需要时取出梨状腺，这个组织是非常粘，往往会坚持您的镊子。当这个程序是正确的，它有可能获得高度纯化的方式从单一的蜘蛛丝绸生产的全部七个腺体。通常情况下，可以恢复微克量的总RNA和蛋白质从一个单一的蜘蛛清扫。例如，使用qPCR的总RNA研究的一个tubuliform限制蛋白基因，TuSp1的mRNA水平（图4）所示。代表实验，涉及蛋白质裂解液从收集到的腺体，如质谱分析（MS / MS分析也可以使用）或氨基酸组成分析显示（图5-6），分别在解决方案的胰蛋白酶消化。 <img src="/files/ftp_upload/2382/2382fig1a.jpg" alt="图1a" /><img src="/files/ftp_upload/2382/2382fig1e.jpg" alt="图1e" /> 图1处理，麻醉，并去除从一个雌性黑寡妇蜘蛛腹部的外骨骼。一）转移到衬塑一个纸板箱，以方便移动到一个较小的塑料瓶蜘蛛的蜘蛛。二）蜘蛛放入一个塑料瓶，带插头。三）与二氧化碳气体麻醉的蜘蛛。 D）分离的头胸部从使用microscissors的腹部。五）固定在腹部解剖托盘使用驱虫引脚。 F）的后外侧切口，用镊子pealed用剪刀和外骨骼的一部分。 G）的切口垂直最初的横向切割。 H）去除脂肪层的外骨骼和曝光。 <img src="/files/ftp_upload/2382/2382fig2.jpg" alt="图2" /> 图2。七个不同的丝绸生产腺体，而在腹部的蜘蛛以及周围组织完好的可视化。一）图片的tubuliform，主要ampullate，flagelliform，汇总，在12倍的放大倍率轻微ampullate组织。二）12X放大倍率aciniform和梨状腺体的图像。 <img src="/files/ftp_upload/2382/2382fig3a.jpg" alt="图3a" /><img src="/files/ftp_upload/2382/2382fig3e.jpg" alt="图3E" /> 图3的照片后，他们从腹部去除丝绸生产的七个腺体。除aciniform和梨状腺体，这是在40X与20X放大倍率的所有图像被抓获。 a）主要ampullate腺体;二）主要和次要ampullate（右侧）的大小比较;三）tubuliform，D）flagelliform，E）聚合; F）的aciniform，G）梨形。 <img src="/files/ftp_upload/2382/2382fig4.jpg" alt="图4" /> 丝的基因，TuSp1，调查后，在不同的丝绸生产腺体TuSp1 mRNA水平从使用实时定量PCR（定量PCR）的总RNA以下隔离（不包括梨状腺）的表达模式的代表结果图4。腺体。 <img src="/files/ftp_upload/2382/2382fig5.jpg" alt="图5" /> 图5：从梨状腺取得的下列解决方案中的胰蛋白酶消化的蛋白提取物的质谱分析的例子。注：X2代表谱线强度的2倍放大倍率。 ＃符号显示频谱肽离子对应PySp1素地区的群众。 <img src="/files/ftp_upload/2382/2382fig6.jpg" alt="图6" /> 图6：从tubuliform腺体中提取的蛋白质的氨基酸组成轮廓的典型结果。注：ASX = ASP和ASN; GLX = Glu和Gln。蓝着色反映与极性侧链组的氨基酸，而红色着色代表与非极性侧链组的氨基酸残基。

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我们从黑寡妇蜘蛛丝生产腺体的显微解剖的方法提供了一种有效的手段，获得高度纯化的的丝绸生产的腺体。解剖在1.5至3小时就可以完成，产生了七个不同的一套完整的丝绸生产从cobweavers腺体。获得高度纯化的样品丝腺，让调查人员能够执行广泛的生化研究，包括确定新的丝绸或使用质谱分析mRNA水平的选择性表达的基因在不同的腺体内腺体管腔内容存储伴侣蛋白，并在体外培养特定腺体?...

<table><tbody><tr><td>Sodium chloride</td><td>Electron Microscopy Sciences</td><td>SX0420-1</td><td>0.1 M in water</td></tr><tr><td>Diethyl pyrocarbonate</td><td>Sigma-Aldrich</td><td>D-5758 - 5 ml</td><td>0.1% v/v</td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>S1804 - 1 kg</td><td>0.015 M in water</td></tr><tr><td>Dissecting microscope</td><td>Leica Microsystems</td><td>Leica MZ16</td><td></td></tr><tr><td>Digital microscope camera</td><td>Leica Microsystems</td><td>DFC320</td><td>Software - Leica Application Suite v2.8.1</td></tr><tr><td>Vannas scissors</td><td>World Precision Instruments, Inc.</td><td>500260</td><td></td></tr><tr><td>Stainless steel forceps</td><td>World Precision Instruments, Inc.</td><td>501764</td><td>Mini Dumont #M5S</td></tr><tr><td>Insect pins</td><td>Indigo Instruments</td><td>33414-2</td><td>Insect pins #2</td></tr><tr><td>Small or large dissection dishes</td><td>Living Systems Instrumentation</td><td>DD-50-S or DD-90-S</td><td>52 mm diameter x 18 mm H (Sylgard Depth ~6mm) or 93 mm x 22 mm</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; culture vials</td><td>Carolina Biological</td><td>FR-17-3076</td><td>Size is 31.75 mm diameter x 101.6 mm</td></tr></tbody></table>

microdissection of black widow spider silk-producing glands

现代蜘蛛自旋高性能纤维丝，具有广泛的生物学功能，包括运动，捕获猎物和保护发展的后代 1,2 。蜘蛛来完成这些任务的纺纱几个不同的光纤类型有不同的力学性能。这种专业化的光纤类型有发生，通过不同的丝腺，小biofactories功能的演变。这些biofactories制造和储存大量的丝蛋白纤维生产。通过一系列复杂的生化事件，这些丝蛋白是从液态转换成固体物质一经挤出。 机械的研究表明，蜘蛛丝都强于高强度钢3。了解蜘蛛丝线的结构和功能之间的关系的分析发现，蜘蛛丝主要包括蛋白质，或fibroins，在其蛋白质序列4块重复。通过翻译的丝绸的cDNA分析，作出贡献，以令人难以置信的蜘蛛丝的拉伸强度和可扩展性的分子共同签名正在揭开。由于蜘蛛丝的非凡的材料特性，世界各地的研究实验室都在竞相理解和模仿的纺纱工艺，生产人造丝纤维，商业，军事和工业应用。纺纱的研究实验室中的人造蜘蛛丝的主要挑战之一，涉及了从丝绸生产腺体的纤维的挤压过程中发生的生化过程的完整的理解。 在这里，我们提出了七个不同的丝绸生产cobweaving黑寡妇蜘蛛，其中包括主要和次要的ampullate腺体腺体[生产吊斗铲和脚手架丝绸] 5,6，tubuliform [综合蛋的情况下丝] 7隔离的方法，8，flagelliform [COB -织布未知功能]，总[胶丝，aciniform [综合猎物包装和蛋的情况下线程] 9号和梨状[生产附件光盘丝绸] 10。这种方法是根据麻醉蜘蛛与二氧化碳气体，随后的分离从腹部的头胸部和腹部的显微切割获得的丝绸生产的腺体。随着丝绸生产的不同腺体分离，这些组织可用于检索不同的大分子，包括实时定量PCR，北部，免疫印迹，质谱（MS或MS / MS）分析，确定不同的生化分析新的蚕丝蛋白序列，蛋白质参与丝绸大会通路，或使用完整的组织细胞培养或组织学实验搜索。

Watch this Scientific Journal Video about Microdissection of Black Widow Spider Silk-producing Glands at JoVE.com

Microdissection of Black Widow Spider Silk-producing Glands

在这里，我们描述了一个有效的策略，以消除雌性黑寡妇蜘蛛腹部的丝生产腺体。此过程允许的七个不同的丝绸生产腺体高度纯化的时尚，一个调查研究蜘蛛丝的生产和纤维大会的重要的过程中迅速隔离。

显微切割的黑寡妇蜘蛛丝绸生产腺体

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

microdissection-of-black-widow-spider-silk-producing-glands

Research

JoVE Journal

Biology

15.7K 次观看.  University of the Pacific. 在这里，我们描述了一个有效的策略，以消除雌性黑寡妇蜘蛛腹部的丝生产腺体。此过程允许的七个不同的丝绸生产腺体高度纯化的时尚，一个调查研究蜘蛛丝的生产和纤维大会的重要的过程中迅速隔离。

视频: Microdissection of Black Widow Spider Silk-producing Glands

Synthetic Spider Silk Production on a Laboratory Scale

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation biomaterials with high performance properties. The development of these new structural materials must be rapid, cost-efficient and involve processing methodologies and products that are environmentally friendly and sustainable. Spiders spin a multitude of different fiber types with diverse mechanical properties, offering a rich source of next generation engineering materials for biomimicry that rival the best manmade and natural materials. Since the collection of large quantities of natural spider silk is impractical, synthetic silk production has the ability to provide scientists with access to an unlimited supply of threads. Therefore, if the spinning process can be streamlined and perfected, artificial spider fibers have the potential use for a broad range of applications ranging from body armor, surgical sutures, ropes and cables, tires, strings for musical instruments, and composites for aviation and aerospace technology. In order to advance the synthetic silk production process and to yield fibers that display low variance in their material properties from spin to spin, we developed a wet-spinning protocol that integrates expression of recombinant spider silk proteins in bacteria, purification and concentration of the proteins, followed by fiber extrusion and a mechanical post-spin treatment. This is the first visual representation that reveals a step-by-step process to spin and analyze artificial silk fibers on a laboratory scale. It also provides details to minimize the introduction of variability among fibers spun from the same spinning dope. Collectively, these methods will propel the process of artificial silk production, leading to higher quality fibers that surpass natural spider silks.

Despite the outstanding mechanical and biochemical properties of spider silks, this material cannot be harvested in large quantities by conventional means. Here we describe an efficient strategy to spin artificial spider silk fibers, which is an important process for investigators studying spider silk production and their use as next-generation biomaterials.

实验室规模的人工合成蜘蛛丝生产

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

科研

生物工程

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

环境科学

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee (Apis mellifera) Workers

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired glands that are located in the head of the bee are highly sensitive to the quantity and quality of pollen and pollen substitutes that the nurse bee consumes. The glands get smaller when nurses are fed deficient diets and are large when they are fed complete diets. Because nurse hypopharyngeal gland size is a robust indicator of nurse nutrition, it is essential that those studying honey bee nutrition know how to measure these glands. Here, we provide detailed methods for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal glands. We present comparisons of unstained and stained tissue and data that were used to study the impact of pollen on gland size. This method has been used to test how diet impacts hypopharyngeal gland size but has further use for understanding the role of these glands in hive health.

Measuring Hypopharyngeal Gland Acinus Size in Honey Bee Apis mellifera Workers

Hypopharyngeal gland acinus size is a robust measure of nurse honey bee nutrition. Here, we provide a detailed protocol for dissecting, staining, imaging, and measuring nurse bee hypopharyngeal gland acini.

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

The nurse hypopharyngeal glands produce the protein fraction of the worker and royal jelly that is fed to developing larvae and queens. These paired ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

分析小鼠沿着轴突生长的雪旺氏细胞发展

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

神经科学

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

直立活体显微术制备小鼠颌下腺涎腺

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

免疫与感染

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, ...

生物学

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. These structures are almost always localized on plant surfaces. Among them are nectaries or glandular trichomes. The secreted compounds are often employed in interactions with the biotic environment, for example as attractants for pollinators or deterrents against herbivores.
Glands that are unique in several aspects can be found in carnivorous plants. In so-called pitcher plants of the genus Nepenthes, bifunctional glands inside the pitfall-trap on the one hand secrete the digestive fluid, including all enzymes necessary for prey digestion, and on the other hand take-up the released nutrients. Thus, these glands represent an ideal, specialized tissue predestinated to study the underlying molecular, biochemical, and physiological mechanisms of protein secretion and nutrient uptake in plants. Moreover, generally the biosynthesis of secondary compounds produced by many plants equipped with glandular structures could be investigated directly in glands.
In order to work on such specialized structures, they need to be isolated efficiently, fast, metabolically active, and without contamination with other tissues. Therefore, a mechanical micropreparation technique was developed and applied for studies on Nepenthes digestion fluid. Here, a protocol is presented that was used to successfully prepare single bifunctional glands from Nepenthes traps, based on a mechanized microsampling platform. The glands could be isolated and directly used further for gene expression analysis by PCR techniques after preparation of RNA.

Isolation of Viable Multicellular Glands from Tissue of the Carnivorous Plant, Nepenthes

Plants glands are specialized structures responsible for the biosynthesis and secretion of many compounds involved in interactions with the biotic environment. To enable studies on their molecular and biochemical features, a mechanical micropreparation technique was established in order to isolate single metabolically active glands, here from the carnivorous plant Nepenthes.

从食虫植物组织分离存活的多细胞腺的，猪笼草</i

Many plants possess specialized structures that are involved in the production and secretion of specific low molecular weight compounds and proteins. ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional ...

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

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 architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

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 ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

冈 比亚按蚊 幼虫唾液腺的解剖和免疫染色

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

显微切割的黑寡妇蜘蛛丝绸生产腺体

摘要

探索更多视频

此视频中的章节

实验室规模的人工合成蜘蛛丝生产

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

分析小鼠沿着轴突生长的雪旺氏细胞发展

直立活体显微术制备小鼠颌下腺涎腺

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

从食虫植物组织分离存活的多细胞腺的，猪笼草

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析

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

冈比亚按蚊幼虫唾液腺的解剖和免疫染色

显微切割的黑寡妇蜘蛛丝绸生产腺体

摘要

探索更多视频

此视频中的章节

实验室规模的人工合成蜘蛛丝生产

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

蜂蜜蜂 (api 蜜蜂) 中下咽腺性腺泡大小的测定

分析小鼠沿着轴突生长的雪旺氏细胞发展

直立活体显微术制备小鼠颌下腺涎腺

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

从食虫植物组织分离存活的多细胞腺的，猪笼草

提取毒液和毒腺Microdissections从蜘蛛的蛋白质组学和转录组分析

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

冈 比亚按蚊 幼虫唾液腺的解剖和免疫染色

冈比亚按蚊幼虫唾液腺的解剖和免疫染色