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。

<ol>
	<li>Vollrath, F., Knight, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Int. J. Biol. Macromol. 24, 243-243 (1999).</li><li>Gosline, J. M., Guerette, P. A., Ortlepp, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Exp. Biol. 202, 3295-3295 (1999).</li><li>Gosline, J. M., DeMont, M. E., Denny, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endeavour. 10, 31-31 (1986).</li><li>Hinman, M. B., Jones, J. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Trends Biotechnol. 18 (9), 374-374 (2000).</li><li>Lewis, R. V., Xu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proc. Natl. Acad. Sci. 87, 7120-7120 (1990).</li><li>Colgin, M. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protein Sci. 7 (3), 667-667 (1998).</li><li>Tian, M., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Appl. Phys. A-Mater. 82, 265-265 (2006).</li><li>Hu, X., Lawrence, B., Kohler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biochemistry. 44 (30), 10020-10020 (2005).</li><li>Vasanthavada, K., Hu, X., Falick, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Biol Chem. 282 (48), 35088-35088 (2007).</li><li>Hayashi, C. Y., Blackledge, T. A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mol. Biol. Evol.. 21 (10), 1950-1950 (2004).</li><li>Blasingame, E., Tuton-Blasingame, T., Larkin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J Biol Chem. 284 (42), 29097-29097 (2009).</li></ol>

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?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Sodium chloride</td>
<td>&nbsp;</td>
<td>EM Science</td>
<td>SX0420-1</td>
<td>0.1 M in water</td>
</tr>
<tr>
<td>Diethyl pyrocarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D-5758 - 5 ml</td>
<td>0.1% v/v</td>
</tr>
<tr>
<td>Sodium citrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S1804 - 1 kg</td>
<td>0.015 M in water</td>
</tr>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>Leica MZ16</td>
<td>&nbsp;</td>
<tr>
<td>Digital microscope camera</td>
<td>&nbsp;</td>
<td>Leica Microsystems</td>
<td>DFC320</td>
<td>Software - Leica Application Suite v2.8.1</td>
</tr>
<tr>
<td>Vannas scissors</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500260</td>
<td>&nbsp;</td>
<tr>
<td>Stainless steel forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>501764</td>
<td>Mini Dumont #M5S</td>
</tr>
<tr>
<td>Insect pins</td>
<td>&nbsp;</td>
<td>Indigo Instruments</td>
<td>33414-2</td>
<td>Insect pins #2</td>
</tr>
<tr>
<td>Small or large dissection dishes</td>
<td>&nbsp;</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>Drosophila culture vials</td>
<td>&nbsp;</td>
<td>Carolina Biological Supply Company</td>
<td>FR-17-3076</td>
<td>Size is 31.75 mm diameter x 101.6 mm</td>
</tr>		</tbody>
		</table>
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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

Dissection of Midgut and Salivary Glands from Ae. aegypti Mosquitoes

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol demonstrates dissection techniques for removal of the midgut and salivary glands from Aedes aegypti mosquitoes. 



The mosquito midgut and salivary glands are key entry and exit points for vector pathogens like Plasmodium falciparum and the dengue virus. This video demonstrates the dissection techniques for removing the midgut and salivary glands from Aedes aegypti mosquitoes.

从AE肠和唾液腺的解剖。埃及伊蚊蚊

The mosquito midgut and salivary glands are key entry and exit points for pathogens such as Plasmodium parasites and Dengue viruses. This video protocol ...

科研

生物学

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

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. ...

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

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can also be used to investigate and validate novel techniques and algorithms in volumetric medical imaging and therapies. Creating 3D high-resolution atlases of different organs1,2,3 is another application of histology volume reconstruction. This provides a resource for investigating tissue structures and the spatial relationship between various cellular features. We present an image registration approach for histology volume reconstruction, which uses a set of optical blockface images. The reconstructed histology volume represents a reliable shape of the processed specimen with no propagated post-processing registration error. The Hematoxylin and Eosin (H&#38;E) stained sections of two mouse mammary glands were registered to their corresponding blockface images using boundary points extracted from the edges of the specimen in histology and blockface images. The accuracy of the registration was visually evaluated. The alignment of the macrostructures of the mammary glands was also visually assessed at high resolution.
This study delineates the different steps of this image registration pipeline, ranging from excision of the mammary gland through to 3D histology volume reconstruction. While 2D histology images reveal the structural differences between pairs of sections, 3D histology volume provides the ability to visualize the differences in shape and volume of the mammary glands.

We present an image registration approach for 3-dimensional (3D) histology volume reconstruction, which facilitates the study of the changes of an organ at the level of macrostructures made up of cells . Using this approach, we studied the 3D changes between wild-type and Igfbp7-null mammary glands.

3维组织学音量和其研究小鼠乳腺腺体的应用改造

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

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

灭鼠工作心脏模型心肌的性能和氧耗的研究

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

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 ...

Investigating Teliospore Germination Using Microrespiration Analysis and Microdissection

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are thick-walled dispersal agents. Teliospores can remain dormant for decades. The dormancy is characterized by low metabolic rates, paused macromolecular biosynthesis and greatly reduced levels of respiration. Upon receiving required environmental signals, teliospores germinate to produce haploid cells, which can initiate new rounds of infection. Teliospore germination is characterized by the resumption of macromolecular biosynthesis, increased respiration and dramatic morphological changes. In order to precisely measure changes in cellular respiration during the early stages of germination, we have developed a simple protocol employing a Clark-type respirometer. The later stages of germination are distinguished by specific morphological changes, but germination is asynchronous. We developed a microdissection technique that enables us to collect teliospores at distinct germination stages.

Smut fungi cause many devastating agricultural diseases. They are dispersed as dormant teliospores that germinate in response to environmental cues. We outline two methods to investigate molecular changes during germination: measuring respiration increase to detect metabolic activation and assessing changing molecular events by isolating teliospores at distinct morphological stages.

利用 Microrespiration 分析和显微解剖研究 Teliospore 萌发

Smut fungi are the etiological agents of several devastating agricultural diseases. They are characterized by the production of teliospores, which are ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成年小鼠泪液和下颌腺肌上皮细胞的分离

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...