Name: an in vivo method to study mouse blood-testis barrier integrity
Uploaded: 2018-12-02T13:00:00
Description: Watch this Scientific Journal Video about An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity at JoVE.com

这项工作得到了国家重点研发项目 (2016yfa0500902)、国家自然科学基金 (31471228, 31471228)、江苏杰出青年科学基金会 (bk20150047)、自然科学组织的支持。江苏省成立 (bk20140897, 14kka180005) 和江苏省创新型企业项目到 k. z。

所有进行的动物实验都得到了南京医科大学委员会的批准。雄性 c57bl6 小鼠在光周期条件下保存, 并提供食物和水。
1. 准备工作
<ol><li>
微注射毛细血管
	<ol>
<li>使用外径、内量计和长度分别为 1.0 mm、0.8 mm 和 10.0 cm 的微注射毛细血管。</li>
		<li>用毛细管拉拔玻璃毛细血管 (图 1a)。根据所使用的毛细管牵引机, 测试和调整设置。</li>
		<li>用钳?...

<ol>
	<li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sertoli-Sertoli+and+Sertoli-germ+cell+interactions+and+their+significance+in+germ+cell+movement+in+the+seminiferous+epithelium+during+spermatogenesis.">Sertoli-Sertoli and Sertoli-germ cell interactions and their significance in germ cell movement in the seminiferous epithelium during spermatogenesis.</a> Endocrine Reviews. 25 (5), 747-806 (2004).</li><li>Wen, Q., 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=Transport+of+germ+cells+across+the+seminiferous+epithelium+during+spermatogenesis-the+involvement+of+both+actin-+and+microtubule-based+cytoskeletons.">Transport of germ cells across the seminiferous epithelium during spermatogenesis-the involvement of both actin- and microtubule-based cytoskeletons.</a> Tissue Barriers. 4 (4), e1265042 (2016).</li><li>Wang, C. Q., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+seamless+trespass:+germ+cell+migration+across+the+seminiferous+epithelium+during+spermatogenesis.">A seamless trespass: germ cell migration across the seminiferous epithelium during spermatogenesis.</a> Journal of Cell Biology. 178 (4), 549-556 (2007).</li><li>Fijak, M., Meinhardt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+testis+in+immune+privilege.">The testis in immune privilege.</a> Immunological Reviews. 213, 66-81 (2006).</li><li>O'Bryan, M. K., Hedger, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+Networks+in+the+Control+of+Spermatogenesis+Chronic+Inflammation+in+an+Immunologically+Privileged+Tissue?.">Inflammatory Networks in the Control of Spermatogenesis Chronic Inflammation in an Immunologically Privileged Tissue?.</a> Molecular Mechanisms In Spermatogenesis. 636, 92-114 (2008).</li><li>Li, N., Wang, T., Han, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+cellular+and+molecular+aspects+of+immune+privilege+in+the+testis.">Structural cellular and molecular aspects of immune privilege in the testis.</a> Frontiers in Immunology. 3, 152 (2012).</li><li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mammalian+Blood-Testis+Barrier:+Its+Biology+and+Regulation.">The Mammalian Blood-Testis Barrier: Its Biology and Regulation.</a> Endocrine Review. 36 (5), 564-591 (2015).</li><li>Govero, J., 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=Zika+virus+infection+damages+the+testes+in+mice.">Zika virus infection damages the testes in mice.</a> Nature. 540 (7633), 438-442 (2016).</li><li>Jenabian, M. A., 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=Immune+tolerance+properties+of+the+testicular+tissue+as+a+viral+sanctuary+site+in+ART-treated+HIV-infected+adults.">Immune tolerance properties of the testicular tissue as a viral sanctuary site in ART-treated HIV-infected adults.</a> AIDS. 30 (18), 2777-2786 (2016).</li><li>Holembowski, 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=TAp73+is+essential+for+germ+cell+adhesion+and+maturation+in+testis.">TAp73 is essential for germ cell adhesion and maturation in testis.</a> Journal Of Cell Biology. 204 (7), 1173-1190 (2014).</li><li>Legendre, A., 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=An+engineered+3D+blood-testis+barrier+model+for+the+assessment+of+reproductive+toxicity+potential.">An engineered 3D blood-testis barrier model for the assessment of reproductive toxicity potential.</a> Biomaterials. 31 (16), 4492-4505 (2010).</li><li>Setchell, B. P., Waites, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+permeability+of+the+testicular+capillaries+and+of+the+'blood-testis+barrier'+after+injection+of+cadmium+chloride+in+the+rat.">Changes in the permeability of the testicular capillaries and of the 'blood-testis barrier' after injection of cadmium chloride in the rat.</a> Journal of Endocrinology. 47 (1), 81-86 (1970).</li><li>Griswold, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+central+role+of+Sertoli+cells+in+spermatogenesis.">The central role of Sertoli cells in spermatogenesis.</a> Seminars in Cell &amp; Developmental Biology. 9 (4), 411-416 (1998).</li><li>Cheng, C. Y., Mruk, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-testis+barrier+and+its+implications+for+male+contraception.">The blood-testis barrier and its implications for male contraception.</a> Pharmacological Reviews. 64 (1), 16-64 (2012).</li><li>Mruk, D. D., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+system+to+study+Sertoli+cell+blood-testis+barrier+dynamics.">An in vitro system to study Sertoli cell blood-testis barrier dynamics.</a> Methods Molecular Biology. 763, 237-252 (2011).</li><li>Orth, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+of+Sertoli+cells+in+fetal+and+postnatal+rats:+a+quantitative+autoradiographic+study.">Proliferation of Sertoli cells in fetal and postnatal rats: a quantitative autoradiographic study.</a> Anatomical Record. 203 (4), 485-492 (1982).</li><li>Lee, N. P. Y., Mruk, D., Lee, W. M., Cheng, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+the+cadherin/catenin+complex+a+functional+unit+of+cell-cell+actin-based+adherens+junctions+in+the+rat+testis?.">Is the cadherin/catenin complex a functional unit of cell-cell actin-based adherens junctions in the rat testis?.</a> Biology of Reproduction. 68 (2), 489-508 (2003).</li><li>Bai, S., 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=A+Germline-Specific+Role+for+the+mTORC2+Component+Rictor+in+Maintaining+Spermatogonial+Differentiation+and+Intercellular+Adhesion+in+Mouse+Testis.">A Germline-Specific Role for the mTORC2 Component Rictor in Maintaining Spermatogonial Differentiation and Intercellular Adhesion in Mouse Testis.</a> Molecular Human Reproduction. 24 (5), 244-259 (2018).</li><li>Korhonen, H. M., 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=DICER+Regulates+the+Formation+and+Maintenance+of+Cell-Cell+Junctions+in+the+Mouse+Seminiferous+Epithelium.">DICER Regulates the Formation and Maintenance of Cell-Cell Junctions in the Mouse Seminiferous Epithelium.</a> Biology of Reproduction. 93 (6), 139 (2015).</li><li>Loir, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trout+Sertoli+cells+and+germ+cells+in+primary+culture:+I.+Morphological+and+ultrastructural+study.">Trout Sertoli cells and germ cells in primary culture: I. Morphological and ultrastructural study.</a> Gamete Research. 24 (2), 151-169 (1989).</li><li>Chen, H., 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=Monitoring+the+Integrity+of+the+Blood-Testis+Barrier+(BTB):+An+In+Vivo+Assay.">Monitoring the Integrity of the Blood-Testis Barrier (BTB): An In Vivo Assay.</a> Methods in Molecular Biology. 1748, 245-252 (2018).</li></ol>

精子发生在精子上皮, 是一个高度有序和动态的过程, 由生殖细胞和体细胞 (如 塞尔托利细胞)控制13。由塞尔托利细胞构建的 Sertoli 结构将精子上皮分为基底和顶端隔间。减数分裂和单倍体生殖细胞的发育发生在形成免疫屏障的顶端隔间 14.
btb 功能可能会受到有毒物质的损害, 也可能会由于与细胞连接形成有关的基因缺陷而受到...

哺乳动物精子发生被认为是一个高度结构化的过程, 包括精原体自我更新和分化, 通过精母细胞成为单倍体精子通过有丝分裂, 减数分裂, 和精子细胞生成, 在此期间戏剧性发生生化和形态变化。发育中的生殖细胞逐渐从精子管的底部向腔内输送。这个过程是由生殖细胞和塞尔托利细胞1,2之间的细胞接触调节的。相邻的塞尔托利细胞形成位于生精小管基部附近的 Sertoli。btb 物理上将上皮分为基底和肾上腺室。在上皮周期的第八-ix 期, 基底隔间的前瘦素精母细胞通过 btb 迁移, 进入内皮隔间 3.因此, btb 的功能是为完成减数分裂和精子发生 4,5,6提供免疫诊断的微环境.与其他仅由紧密连接点 (tj) 组成的血组织屏障 (如血脑屏障) 不同, btb 由四个不同的连接点 (tj、外质化专业、缝隙连接点和中间丝状连接点) 组成。) 塞尔托利细胞之间 1,7。
许多研究使用转基因小鼠、病毒感染和环境毒物来研究 btb 完整性的机制 7,8, 9.btb 破坏会导致精子发生和不孕不育受损。由于 Sertoli 的形成和完整性已被证实受到塞尔托利细胞8之间接触的影响, 基于分离的塞尔托利细胞原代培养的体外模型已被用于 Sertoli 研究。然而, 该模型不能准确地模拟 btb 动态在体内。此外, 没有建立这种与塞尔托利细胞共同培养的方法, 能够反映 Sertoli10、11 的所有相关结构和功能成分。
一般来说, btb 完整性检测通常基于小分子, 如 ez-link sulfo-nhs-lc-biopin 和 fitc 共轭菊粉 (inulin-fitc). 通常情况下, 生物素或 inulin-FITC 从基底隔间的扩散被 btb 结构阻断。因此, 我们能够使用这种方法来评估 btb 损伤与对照组相比的程度。虽然 btb 可能会受到某些类型的刺激的影响, 例如使用氯化镉 (cdcl2)12的治疗, 但小分子可以接触到 btb, 这些分子最终会作为指标进入肾上腺隔间。
早期体内 btb 完整性检测包括将生物素或 inulin-FITC 注入颈静脉, 这涉及手术, 具有侵入性、复杂性和耗时性。此外, 由于记者的物质通过循环在全身扩散, 在精子管中的生物素或伊努林-fitc 的局部浓度是有限的。此外, 全身接触可能会诱发免疫反应。在这里, 我们提出了一个简单而有效的体内btb 完整性检测, 使直接注射少量的 inulin-FITC 到睾丸间质。使用荧光标记法, 染色过程方便, 因为不需要二级抗体。在这里, 荧光染料进入睾丸的过程是可视化的。

<table border="0" cellpadding="0" cellspacing="0" style="width:803px;" width="802">
	
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Capillary puller&#160;</td>
			<td style="width:231px;">SUTTER INSTRUMENT (USA)</td>
			<td style="width:139px;">P-97</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">10x PBS</td>
			<td style="width:231px;">Hyclone (USA)</td>
			<td style="width:139px;">SH30258.01</td>
			<td>dilution to 1&#215; in ddH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">F6057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesion microscope slides</td>
			<td style="width:231px;">CITOGLAS (China)</td>
			<td style="width:139px;">80312-3161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cadmium chloride</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">655198-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td style="width:231px;">Zeiss (Germany)</td>
			<td style="width:139px;">LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dust-free paper</td>
			<td style="width:231px;">Kimberly-Clark (USA)</td>
			<td style="width:139px;">34120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Inulin-FITC</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">F3272</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microinjection capillaries</td>
			<td style="width:231px;">Zhengtianyi (China)</td>
			<td style="width:139px;">BJ-40</td>
			<td>1.0 mm &#215; 0.8 mm&#160; &#215; 100 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette beveler</td>
			<td style="width:231px;">NARISHIGE (JAPAN)</td>
			<td style="width:139px;">EG-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT</td>
			<td style="width:231px;">SAKURA (JAPAN)</td>
			<td style="width:139px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:231px;">Sigma (USA)</td>
			<td style="width:139px;">P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pentobarbital sodium</td>
			<td style="width:231px;">Merck (Germany)</td>
			<td style="width:139px;">P11011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Shaver&#160;</td>
			<td style="width:231px;">Yashen (China)</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Stereo microscope</td>
			<td style="width:231px;">Nikon (JAPAN)</td>
			<td style="width:139px;">SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose&#160;</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A610498</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Surgical instruments</td>
			<td style="width:231px;">Stronger (China)</td>
			<td style="width:139px;"></td>
			<td>scissors, forceps, needle holder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe</td>
			<td style="width:231px;">KDL (China)</td>
			<td style="width:139px;">20163150518</td>
			<td>0.45 mm &#215; 0.16 mm RW LB</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">thermostatic heater</td>
			<td style="width:231px;">KELL (Nanjing, China)</td>
			<td style="width:139px;">KEL-2010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td >10x TBS, pH 7.6</td>
<td></td>
<td></td>
<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M Tris</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A600194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">1.37 M Nacl</td>
			<td style="width:231px;">Sangon Biotech (China)</td>
			<td style="width:139px;">A610476</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo method to study mouse blood-testis barrier integrity

精子发生是精原体在睾丸生精小管中发育为成熟精子的过程。这一过程得到了血美屏障 (Sertoli) 塞尔托利细胞连接点的支持, Sertoli 是哺乳动物体内最严密的组织屏障, 并将精原细胞上皮分离成两个隔间, 一个是基部和一个阴囊。btb 为减数分裂 i变 i变 i变的生殖细胞创造了一个独特的微环境, 并通过精子发生将减数分裂后的精子体发育为精子。在这里, 我们描述了一种可靠的检测方法来监测小鼠睾丸在体内的完整性。一个完整的 btb 阻止了 fitc 共轭菊粉从基部扩散到精子管的顶端室。与替代方法相比, 此技术适用于研究可能影响 btb 功能或完整性的基因候选物、病毒或环境毒物, 操作简单, 对手术技能的要求极少。

执行 btb 完整性分析的实验设置如图 1所示。用毛细管拉拔和微型移液器斜面拉和锐化微注射毛细血管 (图 1 a和1c)。微注射用恒温加热器和设备如图 1 b 和1 d 所示。
图 2显示了注入 inulin-FITC 的一些关键步骤。在小...

Watch this Scientific Journal Video about An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity at JoVE.com

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

在这里, 我们提出了一个协议, 通过在睾丸中注入 inulin-fitc 来评估血液睾丸屏障的完整性。这是一种有效的体内方法, 研究血液睾丸屏障完整性, 可由遗传和环境元素损害。

小鼠血睾丸屏障完整性的一种活体研究方法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

此方法可以帮助回答生物学领域血液测试屏障中有关基因候选者、病毒或环境毒素的关键问题，这些问题可能会影响血液测试屏障功能或完整性。手术前三天，每天至少治疗三只八周大的C57Black/6雄性小鼠，通过内丙酮注射，每公斤体重5毫克的氯化镉。手术当天，在75%乙醇消毒手术区域放置一个干净的组织，并设置一个恒温加热器到37摄氏度。将麻醉小鼠放在组织上，然后确认对手趾捏没有反应。用剃须刀剃腹部毛，用75%乙醇对裸露的皮肤进行消毒。在预腺体上方做一厘米的皮肤切口，露出腹壁，用小钳子抬起皮肤，在腹膜上做0.5厘米切口。使用钳子搜索脂肪垫周围的表皮和睾丸，小心地拉出脂肪垫，露出一个附加的睾丸。将一张九厘米的纸放在脂肪垫和睾丸下面，用新鲜准备好的 inulin-FITC 溶液装载微注射移液器。将移液器连接到微操纵装置，并使用解剖显微镜轻轻地将微注射移液器尖端插入图尼卡 albuginea 下。缓慢旋转操作杆，将 20 微升的 inulin-FITC 送入睾丸的中间体。如果交付成功，睾丸将变成明亮的绿黄色。立即将睾丸返回到腹腔，用 20 微升 PBS 注射反侧睾丸，然后用手术缝合线关闭皮肤。将鼠标放在恒温加热器的热垫上。注射40分钟后，用锋利的剪刀从每只鼠标中收集睾丸，并在一毫升冰冷的PBS中清洗组织。接下来，在4摄氏度下将睾丸固定在4%的半成甲醛中，12至24小时，然后用1.5毫升洗涤，每次洗涤1%PBS。30%蔗糖脱水过夜后，将样品转移到单独的嵌入框架中，用最佳的切削温度化合物覆盖组织。将低温恒温设置为负 20 摄氏度，并在低温恒温器中放置框架约 10 分钟，直到 OCT 冻结。然后，用新鲜的 OCT 填充每个嵌入框架，直到每个模具中覆盖整个睾丸。当 OCT 的第二层冻结时，获取每个睾丸的五微米厚的横截面，在获得时将组织部分捕获到玻璃显微镜幻灯片上。要对样品进行成像，请先在室温下将幻灯片加热到加湿盒中。10 分钟后，每次洗涤时用新鲜的 Tris 缓冲盐水洗三次幻灯片，让部分干燥 5 分钟，防止光线。使用无尘纸去除任何残留的盐水，并使用 DAPI 补充安装介质安装样品。然后，用倒置盖滑盖住每个样品，并在共和显微镜的 20 倍目标下对样品进行成像。三天的氯化镉处理对血性屏障造成损害，使内蛋白-FITC从基底室扩散到半硝基的层室。相比之下，这种扩散在控制PBS处理的睾丸中被阻断。血睾丸屏障损伤的程度通过计算因努林-FITC扩散距离与同一半尼弗管的半径之比确定。此外，在异质裂解小鼠中，完整的血睾丸屏障阻断通过屏障扩散到细胞腔室，而有条件的 Rictor 敲除小鼠具有受损的血睾丸屏障，允许注射素扩散。在尝试此程序时，重要的是要记住在实验当天准备的 inulin-FITC，并保持组织免受光，因为 FITC 是一个光敏分子。该技术的影响延伸到精子生成受损的治疗，因为血睾丸屏障的功能是提供一个免疫特权的微环境，完成精子生成中的美病。该技术在开发后，为生殖医学领域的研究人员探索小鼠的亚育不育和不孕症提供了途径。不要忘记，使用甲状醛可能非常危险，执行此程序时应始终采取预防措施，如穿着衣服和面罩。

Research

JoVE Journal

Developmental Biology

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

运用<em&gt;离体</em&gt;全胎儿各器官的直立滴文化的过程中鼠标研究器官发育过程

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

An Efficient Method to Obtain Dedifferentiated Fat Cells

一个有效的方法获取反分化脂肪细胞

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

A Hyperandrogenic Mouse Model to Study Polycystic Ovary Syndrome

Hyperandrogenic 小鼠模型研究多囊卵巢综合征

Hyperandrogenemia plays a critical role in reproductive and metabolic function in females and is the hallmark of polycystic ovary syndrome. Developing a ...