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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

17.1K 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

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

小鼠海马组织的前子宫内电和器官切片文化

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

科研

发育生物学

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

住发展拟南芥花的共焦成像

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

小鼠胚胎肾的解剖与培养

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...