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. </li>
	<li> 将前爪和 hindpaws 轻轻地贴在一个能被固定在一个化学油烟罩内的工作表面上 ( 即 " 发泡胶"), 将鼠标固定在仰卧位 (背面与脸向上). </li>
	<li> 执行 transcardial 的灌注. 
	<ol> <li> 用外科剪刀切开小鼠胸腔以暴露心脏 (和其他胸腔器官). </li>
		<li> 用钝钳固定跳动的心脏, 并在左心室插入一个 26G (0.45 毫米) 针。针是附着在10毫升注射器充满素磷酸盐缓冲盐水 (PBS; 补充5毫克/毫升亚硝酸钠和 10 U/毫升肝素, pH 7.4, 温暖到37和 #176; 使用前)。立即用细剪刀切割右心房, 并开始灌注身体与温暖的 PBS, 直到液体退出右心房是明确的. 
		注意: 对于成年小鼠和 10-20 毫升 pbs, 需要大约 5-10 毫升 pbs. </li>
		<li> 保持针到位, 并更换为4% 甲醛 (粉煤灰; pH 值 7.4) 的注射器。分别为新生儿和 P21 小鼠灌注10毫升和20毫升的定影液. 
		注意: 粉煤灰是有毒的。它可能导致皮肤和呼吸道过敏, 眼睛受损。戴上手套, 在化学罩下工作. </li>
	</ol> </li> <li> 斩首固定的鼠标并用剪刀切开头骨骨. 
	<ol> <li> 用小镊子轻轻地将后脑从头骨底部抬起。在蝶鞍鞍的第一个标志, 停止举起, 但持有后脑, 切割垂体柄和神经纤维连接到大脑的底部与罚款剪刀。这一步是至关重要的, 以确保脑垂体是没有解除与大脑. </li>
		<li> 然后继续抬起大脑, 取出整个大脑, 使脑垂体完全暴露。剪下整个蝶鞍区, 包括垂体、外侧三叉神经、和剪刀下的蝶骨 (大约 3 x 和 #160; 5 mm 2 ). </li>
	</ol> </li> <li> 将解剖的组织放入35毫米的盘中, 或将6个井板中的一个包含2毫升冷4% 的粉煤灰 (pH 7.4)。将组织固定在4和 #176; C 为40分钟为 P0 P7 垂体, 1 小时为 P14 垂体, 1.5 h 为 P21-P28 垂体, 和 3 h 为成人垂体. </li>
	<li> 垂体的进一步离解 <ol> <li> 用10毫升 PBS 洗涤固定组织, 5 个变化, 每15分钟。新生儿垂体与周围组织和蝶骨下方的结合, 可直接处理为脱水。然而, 5 天以上的小鼠垂体腺应在立体显微镜下进一步分离. </li>
		<li> 将固定组织放入含有1毫升 PBS 的 35 mm 盘中。对于 P5-P14 垂体, 用细钳和剪刀将神经和骨骼之间的结缔组织膜移除, 并仔细地将脑垂体与侧三叉神经分开, 但离开蝶鞍鞍。孤立的腺体和神经通过连接膜与 #34; H 和 #34; 类似的外观 ( 图 1B ). </li>
		<li> 对于 P21 和成人垂体, 移除垂体周围的神经和结缔组织膜, 并从周围组织中释放腺体。用晶状体清洁组织纸包裹脑垂体组织, 并将其放入嵌入盒中. 
		注: 用晶状体清洁纸巾包裹小脑垂体有助于防止任何潜在的标本丢失, 并在下面的石蜡植入过程中保持组织完整性。它可能没有必要包装的脑垂体, 如果它是在一个小瓶子处理冷冻嵌入程序. </li>
	</ol> </li> </ol> 2。石蜡嵌入和切片 <ol> <li> 在嵌入的磁带中脱水标本, 其中50%、65%、75% 和95% 乙醇溶液为15分钟。随后脱水以纯净的乙醇的3变动, 3 min 每个为 P0-P4 的腺体, 4 min 每 P5-P14 腺体和 5 min 每个为 P21 和更旧的腺体。与二甲苯清除, 3 变化, 3 分钟每 P0 P4 腺体, 4 分钟每 P5 P14 腺体, 5 分钟每 P21 和更老的腺体。渗透与熔融石蜡, 3 变化, 7 分钟, 每 P0-P4 腺体, 8 分钟, 其次是6分钟, 一次为 P5 和老腺体. 
	注: 组织加工的最佳参数可根据经验进行调整。要获得高质量的切片, 应避免不充分或过度脱水。用二甲苯清除组织也是如此. </li>
	<li> 方向当在组织嵌入控制台系统上嵌入 <ol> <li> 时, 将标本从卡带中取出, 并将其放入带有熔融石蜡的基模半中。正确定位的嵌入垂体腺是必不可少的满足冠状切面. </li>
		<li> 对于 P21 和成人脑垂体, 将脑垂体与其短轴垂直于基底模的底面 (剖面平面) 定位。分别使用蝶骨和三叉神经作为 P0-P4 和 P5-P14 垂体的解剖标志。定向标本与他们的三叉神经或横向椎板的蝶骨垂直于底部表面的基础模具. </li>
		<li> 用加热好的镊子轻轻地将组织置于所需位置, 直到蜡变成半固态的冷却盘。用熔化的石蜡把模具顶起来。允许石蜡冷却, 直到蜡完全凝固. 
		注意: 在胚胎日 18.5 (E18.5) 的垂体可以用与新生儿垂体相同的方式治疗。但垂体 (拉特克和 #39; s 袋) 比 E16.5 年轻, 应嵌入整个头部, 并以矢状轴 (从口到枕) 垂直于基本模具的底面. </li>
	</ol> </li> <li> 在-20 和 #176 上冷却石蜡块; C 为 10-20 min, 然后将其切成薄片 (4 和 #181; m 厚度是首选) 与切片。为获得满意的冠状切面, 在切片期间微调石蜡块的位置。在下面的过程中, 将组织切片安装到赖氨酸涂层的玻璃幻灯片上, 以防止切片脱落. 
	注: 另一种方法是, 在4和 #176 的蔗糖溶液 (PBS) 中, 试样可以在15%、30% (W/V) 中脱水, 直到它们沉到底部, 然后用相同的方向法嵌入到低温埋入化合物中, 然后在干冰中快速冷冻。然后将冰冻的组织块切成 8-10 和 #181 的切片; m 使用低温。然而, 冰冻切片的形态通常不如石蜡切片. </li>
</ol> 3。H 和 #38; E 染色和免疫荧光标记 <ol> <li> 在55和 #176 的热块上预热幻灯片; C 和脱与二甲苯, 2 变化, 每4分钟。用95% 乙醇两次和75% 乙醇两次脱水样品, 每份3分钟。用蒸馏水在振动筛上洗5分钟的幻灯片. </li>
	<li> 用于 H 和 #38; E 染色, 在明矾苏木精溶液中染色切片6分钟, 冲洗奔跑自来水为2分钟, 区分与0.2% 氨水为四十五年代, 和 re-rinse 在自来水中2分钟。然后将95% 乙醇用于1分钟的脱水切片, 以曙红为2分钟, 依次脱水、清除和装入. </li>
	<li> 免疫荧光标记 <ol> <li> 在室温下对含有 3% H 2 O 2 和 0.01% NaN 3 的部分进行处理, 使内生过氧化物。在一个高压锅中用沸腾的部分 (1 毫米 EDTA 和1毫米三盐酸, pH 7.4) 在一个压力锅中提取抗原2分钟, 用阻塞缓冲液 (PBS 中5% 山羊血清) 在30和 #176 中孵育. </li>
		<li> 用兔 anti-growth 激素 (GH) 抗体 (1:2、000) 或兔 anti-glial 纤维酸性蛋白 (胶质) 抗体 (1:2, 000) 在阻断缓冲液中过夜, 在4和 #176; C. </li>
		<li> 孵育部分与二次抗体 (山羊兔 IgG-HRP, 1:300 在阻拦缓冲) 为 35 min 在37和 #176; C. </li>
		<li> 在室温下使用 Biotinyl Tyramide (1:50 在含有 0.3% H 2 O 2 的三缓冲盐水中放大信号, 并以 Streptavidin-Alexa594 或 15 (488 在阻塞缓冲器中, 1:300 min, 30 和 #176 C) 进行可视化. </li>
		<li> Counterstain DAPI (PBS 中的50毫克/升) 在室温下为5分钟. 
		注意: 使用 Tyramide 信号放大 (TSA) 系统可能不需要放大信号。或者, 一个荧光共轭的二次抗体可以用来可视化信号. </li>
	</ol> </li> <li> 使用带有 DAPI、德州红和 FITC 过滤器集的荧光显微镜获取图像, #215; 4/0.13 至 #215; 40/0.75 目标和5.0 像素摄像头。使用 360, 488, 594 nm 激光波长成像 DAPI, alexa 488-, 和 alexa 594 染色部分分别。优化的激光功率 (0.8 一般使用) 和曝光时间达到所需的水平为每个通道。在图像采集软件的控制下捕获图像, 然后使用图像处理软件叠加荧光图像. </li>
</ol>

<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>

作者没有什么可透露的。

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。

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

dissection-coronal-slice-preparation-developing-mouse-pituitary

Research

JoVE Journal

Developmental Biology

17.2K 次观看.  Second Military Medical University. 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。

视频: Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

评定神经连接的形成在共培养体系的光遗传学方法

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

科研

发育生物学

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

神经科学

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Modification of a Colliculo-thalamocortical Mouse Brain Slice, Incorporating 3-D printing of Chamber Components and Multi-scale Optical Imaging

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study these two systems and how they interact has been with the use of in vivo preparations. Major advances have been made with acute brain slices containing the thalamocortical and cortico-thalamic pathways in the somatosensory, visual, and auditory systems. With key refinements to our recent modification of the auditory thalamocortical slice1, we are able to more reliably capture the projections between most of the major auditory midbrain and forebrain structures: the inferior colliculus (IC), medial geniculate body (MGB), thalamic reticular nucleus (TRN), and the auditory cortex (AC). With portions of all these connections retained, we are able to answer detailed questions that complement the questions that can be answered with in vivo preparations. The use of flavoprotein autofluorescence imaging enables us to rapidly assess connectivity in any given slice and guide the ensuing experiment. Using this slice in conjunction with recording and imaging techniques, we are now better equipped to understand how information processing occurs at each point in the auditory forebrain as information ascends to the cortex, and the impact of descending cortical modulation. 3-D printing to build slice chamber components permits double-sided perfusion and broad access to networks within the slice and maintains the widespread connections key to fully utilizing this preparation.

Using multiple angles to cut the mouse pup brain, we improve upon a previously-described acute brain slice which captures the connections between most of the major auditory midbrain and forebrain structures.

修改一个Colliculo  - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

The ability of the brain to process sensory information relies on both ascending and descending sets of projections. Until recently, the only way to study ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

医学

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including proliferation, self-renewal and multipotency. In the postnatal and adult brain, NSPCs are mainly present in two neurogenic niches: the subventricular zone (SVZ) lining the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus (DG). The isolation of the neurogenic niches from postnatal brain allows obtaining a higher amount of NSPCs in culture with a consequent advantage of higher yields. The close contact between cells within each neurosphere creates a microenvironment that may resemble neurogenic niches. Here, we describe, in detail, how to generate SVZ- and DG-derived neurosphere cultures from 1&#8722;3-day-old (P1&#8722;3) mice, as well as passaging, for neurosphere expansion. This is an advantageous approach since the neurosphere assay allows a fast generation of NSPC clones (6&#8722;12 days) and contributes to a significant reduction in the number of animal usage. By plating neurospheres in differentiative conditions, we can obtain a pseudomonolayer of cells composed of NSPCs and differentiated cells of different neural lineages (neurons, astrocytes and oligodendrocytes) allowing the study of the actions of intrinsic or extrinsic factors on NSPC proliferation, differentiation, cell survival and neuritogenesis.

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

产后神经球的分离和扩张 （P1+3） 小鼠神经原性球

The neurosphere assay is an extremely useful in vitro technique for studying the inherent properties of neural stem/progenitor cells (NSPCs) including ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

将小鼠大脑显微切割成功能和解剖学上不同的区域

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

发育小鼠垂体的解剖和冠状切片制备

摘要

探索更多视频

此视频中的章节

评定神经连接的形成在共培养体系的光遗传学方法

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

背腹鼠类内侧嗅皮层组织的调查矢状窦片的制备

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

产后神经球的分离和扩张（P1+3）小鼠神经原性球

将小鼠大脑显微切割成功能和解剖学上不同的区域

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

发育小鼠垂体的解剖和冠状切片制备

摘要

探索更多视频

此视频中的章节

评定神经连接的形成在共培养体系的光遗传学方法

对于高分辨率延时成像神经细胞迁移的一个器官型产后脑切片检测

准备使用一个不连续Percoll蔗糖密度梯度的从小鼠皮质Synaptoneurosomes

背腹鼠​​类内侧嗅皮层组织的调查矢状窦片的制备

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

修改一个Colliculo - 丘脑鼠脑切片，收纳室部件的3-D打印和多尺度光学成像

脑肿瘤/器官切片共培养系统研究肿瘤微环境和靶向药物治疗

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