Name: dissection and coronal slice preparation of developing mouse pituitary gland
Uploaded: 2017-11-16T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

这项工作得到了中国国家自然科学基金 (31201086、31470759和 31671219) 和上海自然科学基金 (12ZR1436900) 的资助。

C57BL/6 鼠在特定的无病原体条件下繁殖。所有动物实验方法都符合第二军医大学动物保育和伦理委员会批准的指导方针. 
 1. 产后发育性垂体的解剖 <ol> <li> 执行麻醉: 将新生小鼠 (P0-P5) 放在被压碎的冰上以诱发体温过低。对于年龄超过5天的小鼠, 注射5% 的氨基甲酸乙酯 (30 和 #181; 体重的腹腔) 以诱导麻醉。评估对尾部/脚趾捏的反应。只有在鼠标对有害刺激没有反应时才进行....

<ol>
	<li>Fauquier, T., Lacampagne, A., Travo, P., Bauer, K., Mollard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hidden+face+of+the+anterior+pituitary.">Hidden face of the anterior pituitary.</a> Trends Endocrinol Metab. 13 (7), 304-309 (2002).</li><li>Haedo, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pituitary+function+by+cytokines.">Regulation of pituitary function by cytokines.</a> Horm Res. 72 (5), 266-274 (2009).</li><li>Zhu, X., Gleiberman, A. S., Rosenfeld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+physiology+of+pituitary+development:+signaling+and+transcriptional+networks.">Molecular physiology of pituitary development: signaling and transcriptional networks.</a> Physiol Rev. 87 (3), 933-963 (2007).</li><li>Bancalari, R. E., Gregory, L. C., McCabe, M. J., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary+gland+development:+an+update.">Pituitary gland development: an update.</a> Endocr Dev. 23, 1-15 (2012).</li><li>Kelberman, D., Rizzoti, K., Lovell-Badge, R., Robinson, I. C., Dattani, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+regulation+of+pituitary+gland+development+in+human+and+mouse.">Genetic regulation of pituitary gland development in human and mouse.</a> Endocr Rev. 30 (7), 790-829 (2009).</li><li>Peker, S., Kurtkaya-Yapicier, O., Kilic, T., Pamir, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microsurgical+anatomy+of+the+lateral+walls+of+the+pituitary+fossa.">Microsurgical anatomy of the lateral walls of the pituitary fossa.</a> Acta Neurochir (Wien). 147 (6), 641-648 (2005).</li><li>Wolpert, S. M., Molitch, M. E., Goldman, J. A., Wood, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size,+shape,+and+appearance+of+the+normal+female+pituitary+gland.">Size, shape, and appearance of the normal female pituitary gland.</a> AJR Am J Roentgenol. 143 (2), 377-381 (1984).</li><li>Carbajo-Perez, E., Watanabe, Y. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+proliferation+in+the+anterior+pituitary+of+the+rat+during+the+postnatal+period.">Cellular proliferation in the anterior pituitary of the rat during the postnatal period.</a> Cell Tissue Res. 261 (2), 333-338 (1990).</li><li>Nantie, L. B., Himes, A. D., Getz, D. R., Raetzman, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+signaling+in+postnatal+pituitary+expansion:+proliferation,+progenitors,+and+cell+specification.">Notch signaling in postnatal pituitary expansion: proliferation, progenitors, and cell specification.</a> Mol Endocrinol. 28 (5), 731-744 (2014).</li><li>Tzou, S. C., Landek-Salgado, M. A., Kimura, H., Caturegli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+mouse+pituitary+immunogen+for+the+induction+of+experimental+autoimmune+hypophysitis.">Preparation of mouse pituitary immunogen for the induction of experimental autoimmune hypophysitis.</a> J Vis Exp. (46), (2010).</li><li>Budry, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Related+pituitary+cell+lineages+develop+into+interdigitated+3D+cell+networks.">Related pituitary cell lineages develop into interdigitated 3D cell networks.</a> Proc Natl Acad Sci U S A. 108 (30), 12515-12520 (2011).</li><li>Wilson, D. B., Wyatt, 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=Histopathology+of+the+pituitary+gland+in+neonatal+little+(lit)+mutant+mice.">Histopathology of the pituitary gland in neonatal little (lit) mutant mice.</a> Histol Histopathol. 7 (3), 451-455 (1992).</li><li>Jonkers, B. W., Sterk, J. C., Wouterlood, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardial+perfusion+fixation+of+the+CNS+by+means+of+a+compressed-air-driven+device.">Transcardial perfusion fixation of the CNS by means of a compressed-air-driven device.</a> J Neurosci Methods. 12 (2), 141-149 (1984).</li><li>Cao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZBTB20+is+required+for+anterior+pituitary+development+and+lactotrope+specification.">ZBTB20 is required for anterior pituitary development and lactotrope specification.</a> Nat Commun. 7, 11121 (2016).</li></ol>

对于发育中的小鼠垂体, 由于其细小而脆弱的特征和独特的解剖特征6,8, 在技术上很难获得合适的冠状切面。因此, 一些研究小组选择 mid-sagittal 切片来分析胚胎和新生儿垂体的形态学11,12。虽然垂体的 mid-sagittal 部分也能够显示前、中、后叶在一个单一的切片, 冠状区是高度优先, 因为它可以显示这三叶的最大?...

脑垂体或垂体是一个重要的内分泌器官分泌荷尔蒙的必要的稳态1,2。从解剖学上来说, 脑垂体是一个由垂体和垂体组成的 "合一" 结构。这些部分有不同的胚胎起源和作用非常不同地。垂体来源于神经外胚层, 分泌催产素和抗利尿激素。垂体起源于拉特克的囊, 并负责释放激素, 包括生长激素, 催乳素, 促甲状腺激素, 卵泡刺激激素, 促黄体激素, 肾上腺皮质激素激素,黑刺激激素3,4,5。
脑垂体在小鼠颅骨的蝶骨 (蝶鞍鞍) 的背表面上, 由一个脆弱的茎附着在大脑的地板上。它由三叉神经和前在侧面围拢由光学交叉6,7。腺体有一个细长的椭圆形, 其长轴与头部垂直。在它的背表面上, 垂体和垂体可以很容易地被标定, 前者占据背内侧区, 后者在侧面和腹上伸展。在产后发育过程中, 垂体的大小在出生后的第一个月内迅速增加8,9。然而, 鼠脑垂体仍然是非常小的大小与平均重量1.9 毫克和轴直径的〜3毫米在成人10, 轴直径 2-2.5 毫米在产后日 21 (P21), 只有 1-1.5 毫米在 P0。
冠状节是首选显示两个垂体和垂体在一个单一的切片的小鼠垂体。然而, 由于其体积极小, 解剖结构独特, 因此需要一些技术技能来获得令人满意的垂体腺的冠状部分。在这篇视频文章中, 我们将展示如何解剖小鼠脑垂体, 并在不同发育阶段制备垂体冠状切面。

<table>
	<tbody>
		<tr>
			<td>Tools/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors-straight</td>
			<td>JinZhong</td>
			<td>J21010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine scissors-strainght</td>
			<td>JinZhong</td>
			<td>WA1010</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>JinZhong</td>
			<td>JD1020</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>RS-5015</td>
			<td>for isolation of the pituitary</td>
		</tr>
		<tr>
			<td>26G (0.45mm) needle</td>
			<td>HongDa</td>
			<td></td>
			<td>for transcardial perfusion</td>
		</tr>
		<tr>
			<td>Syringe (1 mL)</td>
			<td>BD</td>
			<td>300841</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Syringe (10 mL)</td>
			<td>BD</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>35mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Lens cleaning paper</td>
			<td>ShuangQuan</td>
			<td></td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Anatomical microscope</td>
			<td>OLYMPUS</td>
			<td>SZX-ILLB2-200</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Embedding cassette</td>
			<td>Thermo Fisher</td>
			<td>22-272423</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Tissue embedding console system</td>
			<td>KEDEE</td>
			<td>KD-BM11</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Thermo Fisher</td>
			<td>HM315R</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Superfrost-Plus slides</td>
			<td>Thermo Fisher</td>
			<td>22-037-246</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Thermo Fisher</td>
			<td>12-543</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>OLYMPUS</td>
			<td>BH2-RFCA</td>
			<td>can be purchased from other vendors</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urethane</td>
			<td>BBI</td>
			<td>EB0448</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9541</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4.12H2O</td>
			<td>Sigma</td>
			<td>71650</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma</td>
			<td>P2222</td>
			<td>for PBS</td>
		</tr>
		<tr>
			<td>NaNO2</td>
			<td>Sigma</td>
			<td>237213</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection</td>
			<td>SPH</td>
			<td>H31022051</td>
			<td>for perfusion saline</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>SCR</td>
			<td>10009218</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>SCR</td>
			<td>10023418</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Thermo Fisher</td>
			<td>8330</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma</td>
			<td>H9627</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>E4009</td>
			<td>for H&#38;E staining</td>
		</tr>
		<tr>
			<td>rabbit anti growth hormone (GH)</td>
			<td>National Hormone</td>
			<td></td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>antibody</td>
			<td>Pituitary Program</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-mouse GFAP antibody</td>
			<td>Sigma</td>
			<td>G9269</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG, HRP</td>
			<td>Jackson</td>
			<td>111-035-003</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>TSA system</td>
			<td>NEN Life Science Products</td>
			<td>NEL700</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Streptavidin, Alexa Fluor 594</td>
			<td>Thermo Fisher</td>
			<td>S32356</td>
			<td>for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-FITC Alexa Fluor 488</td>
			<td>Thermo Fisher</td>
			<td>A11090</td>
			<td>for immunostaining</td>
		</tr>
	</tbody>
</table>

dissection and coronal slice preparation of developing mouse pituitary gland

脑垂体或垂体是一种重要的内分泌器官, 分泌荷尔蒙对体内稳态至关重要。它由两个腺体组成, 分别具有胚胎的起源和功能--垂体和垂体。发育中的小鼠垂体腺是微小的和微妙的细长椭圆形的形状。冠状部分是首选显示两个垂体和垂体在一个单一的切片的小鼠垂体。
该协议的目的是实现适当的垂体冠状部分与保存完好的组织结构从开发小鼠。在这个协议中, 我们详细地描述了如何解剖和处理脑垂体腺体正确地从开发小鼠。首先, 在解剖前, 用 transcardial 灌注甲醛来固定小鼠。然后应用三种不同的解剖技术, 根据小鼠的年龄, 获得完整的垂体腺。对于胚胎期小鼠 (E) 17.5-18.5 和4天的新生儿, 整个蝶鞍区包括蝶骨、腺体和三叉神经的解剖。对于幼崽的产后天数 (P) 5-14, 垂体腺连接与三叉神经是整体解剖。对于3周以上的小鼠, 脑垂体腺体是从周围组织中仔细解剖的。我们还显示如何在适当的方向上嵌入脑垂体腺体, 利用周围的组织作为地标, 以获得满意的冠状部分。这些方法可用于分析发育小鼠垂体腺的组织学和发育特征。

本协议提出了一种解剖小鼠垂体腺的方法。对新生小鼠, 从颅底解剖了整个蝶鞍区, 包括垂体、三叉神经和蝶骨下方。在过程中 (图 1A) 中, 细小而精致的脑垂体保持完好。对于5天以上的小鼠, 附着在三叉神经外侧的垂体腺则被隔离。从 P7 小鼠中分离出的脑垂体的总结构保持完好 (图 1B)。对于 P21 小鼠, 与新生小鼠相比, 垂体腺的?...

Watch this Scientific Journal Video about Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland at JoVE.com

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

发育小鼠垂体的解剖和冠状切片制备

我们提出了解剖垂体腺和准备垂体冠状区的发展小鼠的协议。

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