这项研究得到了美国国立卫生研究院校内研究计划，NIDCD到MH（DC000088）的部分支持

所有动物实验和程序均按照美国国立神经系统疾病和中风研究所动物护理和使用委员会以及国立卫生研究院国家耳聋和其他沟通障碍研究所批准的协议进行。所有实验方案均由美国国立神经系统疾病和中风研究所动物护理和使用委员会以及美国国立卫生研究院国家耳聋和其他沟通障碍研究所批准。所有方法均按照美国国立神经系统疾病和中风研究所动物护理和使用委员会以及国立卫生研究院国家耳聋和其他沟通障碍研究所的相关指南和规定进行。
1. 动物安乐死
<ol><li>使用C57BL / 6J或 Kcnj10-ZsGreen转基因小鼠，出生后第30 +天（P30 +），重量在15-20g之间。</li>
	<li>使用批准的方案对成年小鼠（P30 岁或以上）实施安乐死（此处为 CO2 窒息）。执行斩首作为次要步骤。 注意：如果进行免疫染色，由于与SV毛细血管中的血液非特异性结合，某些抗体可能具有背景信号。用固定剂进行心脏灌注可以通过从SV毛细血管中去除血液来解决这个问题。但是，大多数抗体可以在没有这种方法的情况下获得良好的结果，并且不会在本协议中涵盖。</li>
</ol>2. 露出骨迷宫
<ol><li>通过喷洒70%乙醇并用纸巾擦拭来清洁鼠标。特别注意头部区域以降低污染风险。通过将皮肤拉向鼻子来去除颅骨上的皮肤。</li>
	<li>使用锋利的剪刀，沿中矢状面分开颅骨，以分隔左右部分。</li>
	<li>从头骨中取出大脑以露出骨迷宫。</li>
</ol>3. 内耳摘除
<ol><li>识别颞骨内的内耳（图2A）并使用#55镊子刮掉颅神经。 注意：用户应使用第二组#55镊子用非惯用手稳定标本。如果避开标本的关键区域（例如，不要压碎耳蜗），则可以在整个解剖过程中用稳定钳抓住标本的不同区域。</li>
	<li>使用#55镊子，解剖大疱和胶囊，将它们与周围的骨骼分离。从内耳表面去除任何残留的软组织。</li>
	<li>将标本转移到含有1x冷PBS（磷酸盐缓冲盐水），pH 7.4的透明组织培养皿中，并置于解剖范围内（图2B）。</li>
</ol>4. SV解剖
注意：通过练习，可以将SV解剖为一个类似于丝带的长片。SV是脆弱的，所以如果它碎成碎片，这些可能会一起储存。或者，这些可以根据轮次（例如，基底，中间，顶端）储存在单独标记的孔中。
<ol><li>如果解剖镜光源可以移动，请将一盏灯放在碟子上方，将第二个光源从侧面平行于解剖表面。这样可以使耳蜗骨下的组织具有更好的对比度。</li>
	<li>使用#55镊子，用前庭部分握住标本，耳蜗朝上。</li>
	<li>使用#55镊子，刺穿顶端的耳蜗骨。 注意：#5 镊子比 #55 厚，可用于骨折，但尺寸/强度的增加会牺牲镊子的细度和操作小组织的能力。考虑使用由inox或dumostar等材料制成的镊子，以避免更脆弱的镊子可能造成的损坏。避免将镊子推到骨头下方太深，以免损坏SV。</li>
	<li>使用#55镊子，沿着顶端转动刮擦（图2C），施加温和的力量以折断外骨层的小块。轻轻提起骨头并将其从侧壁上分离。 注意：将这种解剖视为“从SV中取出所有东西”而不是“从耳蜗中解剖SV”可能会有所帮助。</li>
	<li>继续使用#55镊子从顶端和中间转弯中取出耳蜗骨片（图2D，E）。轻轻向下推侧壁，朝中转撬并取出骨壁碎片，露出顶端和中转的侧壁。</li>
	<li>继续使用#55镊子，轻轻地将顶端转弯侧壁推到一边，露出螺旋神经节。沿外毛细胞层从螺旋神经节分离顶端和中转的侧壁。从耳蜗内取出螺旋神经节碎片。 注意：#55 镊子更小，在操作小而精致的组织时具有优势。应格外小心，避免弯曲或损坏它们。</li>
	<li>为了更好地进入基底转弯的侧壁，请使用#55镊子将耳蜗与颞骨的前庭部分分离。将#55镊子放入圆形和椭圆形窗口，然后向下推至前厅。耳蜗会脱离。切除内耳的前庭部分。</li>
	<li>继续使用#55镊子，现在从中间转弯区域开始去除基底耳蜗骨碎片。轻轻推动骨头下方的侧壁层将其分离。</li>
	<li>通过撬动并轻轻拉动组织来去除侧壁最远的基底部分。该区域的骨头可能太厚，无法在不损坏下面的软组织的情况下去除。</li>
	<li>现在，随着侧壁的基部分离，尽可能多地将侧壁与耳蜗分开。这可以通过沿侧壁追踪#55镊子来完成。轻轻刷骨头和剩余侧壁之间的镊子。如果用户经验丰富，这可以一直到顶点。将侧壁移至新鲜的PBS。 注意：虽然较大的SV碎片可能更容易成像，但鉴于组织的微妙性质，可以拍摄较小的碎片（图2F，G），并且根据大小，也可以用于成像。</li>
	<li>使用#55镊子将侧壁平放。SV 应以侧壁内侧较暗的组织层的形式可见。 注意：用户可能会发现，特别是在靠近顶端的地方，一些SV组织在整个解剖过程中偶然从侧壁上脱落。</li>
	<li>轻轻撬开SV层，使其从基底端的侧壁上分离（图2H）。轻轻推开分离的SV，并将镊子进一步移向侧壁的顶端，如果可能的话，将SV分离为一条长带（图2I）。不要用镊子挤压SV来拉动它。如果组织太脆弱而无法抓住，也可以使用组织勺收集，例如霰粒肿刮匙。 注意：移动SV应始终在光学显微镜下进行，以确保它不会丢失。使用镊子抓握时要小心，因为有损坏的风险。用户可能会发现使用霰粒肿刮匙可降低SV组织受损的风险。对于单核测序，请继续执行第 5 部分。对于 SV 免疫染色，请继续执行第 7 部分。</li>
</ol>5. SV单核悬浮液
注意：本协议适用于SV组织，特别是来自已发布的制造商的单核悬浮方案。可以使用来自不同制造商的平台31.鉴于特定于平台的变化，建议查看设备随附的制造商特定协议。为了达到sNuc-Seq的最佳结果并最大限度地减少RNA降解，组织解剖越快越好（建议在安乐死后15-20分钟内）。一次对一只动物实施安乐死可能会有所帮助，并且仅在准备解剖时才实施安乐死。让多人同时进行解剖也可以消除降解时间（例如，一个实验室人员在左耳工作，而另一个实验室人员在右耳工作）。
<ol><li>如果SV组织将用于sNuc-Seq，请将组织置于冷却的裂解缓冲液中（10 mM Tris-HCl，10 mM NaCl，3 mM MgCl 2,0.005%无核酸酶无核酸酶水溶液）。 注意：如果要在解剖后立即进行测序，请继续执行协议。如果需要暂停实验方案，可以通过将SV放入RNAlater中来保存SV。在4°C下储存过夜，然后在液氮中快速冷冻并在-80°C下储存直至准备使用。准备使用时，在PBS中解冻并洗涤两次，每次5分钟，然后进行均质化（步骤5.2）。</li>
	<li>在 2 mL 弹跳均质器中匀浆组织，在冰上冲程 10-20 次。 注意：玻璃圆筒将手动接触玻璃管的底部，使SV碎片化。SV很精致，因此20冲程通常可以成功实现均质化。</li>
	<li>在冰上裂解组织25分钟。 注意：裂解时间因组织类型而异。在SV组织中裂解25分钟可能会达到低细胞活力（小于4%），但如果细胞活力过高，则可以调整时间（步骤5.8）。</li>
	<li>通过30μm过滤器过滤，并在4°C下以500× g 旋转滤液5分钟。</li>
	<li>除去上清液并将细胞沉淀重悬于 1 mL 细胞核洗涤液和重悬缓冲液（1x PBS、1% 牛血清白蛋白 [BSA]、0.2U/μL RNase 抑制剂）中。</li>
	<li>通过10μm过滤器过滤细胞，并在4°C下以500× g 离心5分钟。</li>
	<li>取出上清液并重悬于 50 μL 细胞核洗涤液和重悬缓冲液中。</li>
	<li>使用细胞计数器和台盼蓝染色计数细胞核。将 5 μL 细胞悬液稀释到 50 μL 1x PBS 中以进行细胞计数;此时的生存能力应该是最小的（3%-4%）。如果发现活力更高，请调整步骤5.3（裂解）的方案并标准化裂解时间以确保细胞充分裂解。 注意：单细胞核制备中的高细胞活力意味着完整细胞比预期的要多，并且可能需要额外的消化。</li>
	<li>将具有所需核密度的样品加载到制造商的设备/芯片上（参见制造商指南）。单核捕获现已准备就绪。 注意：从一只成年小鼠SV中，通常达到50μL浓度为150,000个细胞核/ mL的悬浮液。</li>
</ol>6. SV单核测序
<ol><li>将样品送到核心设施进行测序。 注意：协议的这一部分遵循以下一般步骤：（1）凝胶珠内乳液（GEM）生成和条形码，（2）GEM-RT后净化和cDNA扩增，以及（3）3'基因表达文库构建。sNuc-Seq协议的其余部分相对较长，建议读者使用其制造商特定的协议。制造商指南可能会描述带有特定详细信息和随附图像的步骤。制造商网站上也可能有“操作方法”视频。生成的数据集可以以降维方式表示，例如均匀流形近似和投影（UMAP; 图3）。许多实验室使用测序核心，可以为用户执行此协议。</li>
</ol>7. SV免疫染色和组织安装
<ol><li>如果组织将用于免疫染色，请将其转移到 24 孔板中，浸没在 1x PBS 中的 200 μL 4% 多聚甲醛 （PFA） 中，并在室温下孵育 20 分钟。 注意：在显微镜下进行所有液体去除、洗涤和免疫染色是有帮助的。移液组织时的可视化将确保在去除液体过程中不会意外丢失。只要体积足够大以将SV组织浸没在溶液中，就可以调整抗体和洗涤液的体积。</li>
	<li>固定步骤后，通过在4°C的1x PBS（约500μL）中进行两次短洗涤，每次在低（30-50）RPM的轨道振荡器上5分钟来除去PFA。如果储存，组织可以储存在4°C的1x PBS中。</li>
	<li>取出PBS，在室温下在约300μLPBS-T溶液中透化并封闭至少1小时（0.05吐温20在1x PBS中，含10%胎牛血清）。 注意：一抗和二抗应稀释在此封闭溶液中。</li>
	<li>根据所用特定抗体的规格用一抗染色组织，通常在4°C下过夜。去除剩余的PBS并加入稀释的一抗。 注意：稀释液应根据制造商的建议、公布的数据、以前的经验等进行选择。通常，要测试的第一个浓度是1：100-200稀释度。对于本文中介绍的示例，GS-IB4和DAPI抗体均以1：200稀释。如果对一种以上的蛋白质进行染色，可以将多种一抗组合到同一溶液中，只要每种一抗具有不同的宿主，以避免二抗的交叉反应。</li>
	<li>使用大约 500 μL 的 1x PBS 两次在低 RPM 的轨道振荡器上洗涤组织上的一抗，每次 10 分钟。 注意：继续确保SV组织浸没在溶液中，而不是卡在孔的侧面。</li>
	<li>根据所用特定抗体的规格用二抗染色，通常在室温下在低RPM的轨道振荡器上染色2小时。盖上 24 孔板，使荧光标记的二抗免受光照。 注意：如果需要多个二抗，请将二抗组合到同一溶液中。确保避免交叉标记。</li>
	<li>使用大约 500 μL 的 1x PBS 在低 RPM 的轨道振荡器上洗涤组织上的二抗四次，每次 5 分钟。保持 24 孔板盖住以防止光线照射。</li>
	<li>准备较大的载玻片（75 mm x 25 mm），沿25 mm轴放置两条小胶条，仅比18 mm x 18 mm较小的玻璃盖玻片小。将一滴封片剂（约 50 μL）放在胶条之间。 注意：胶水会产生少量的垂直空间，以便当第二个盖玻片放在顶部时，SV组织样本可以安全地存在，但继续保持在原位。虽然条纹厚度为30-40微米，但应避免挤压玻璃表面之间的组织以保持形态。或者，也可以使用透明胶带。</li>
	<li>使用#55镊子，尽可能少地抓住SV的一端的组织，并从PBS转移到较大的显微镜（75 mm x 25 mm）上的安装试剂滴上。将较小的玻璃盖玻片（18 mm x 18 mm）的一端放在放置一条胶水的载玻片上，轻轻释放以避免产生气泡。盖玻片将落到另一条胶水条纹上，并覆盖SV组织。</li>
	<li>通过在支架的每个角上涂抹一滴透明的指甲油来密封支架。在远离可视化区域的玻璃盖玻片上相应地标记标本。它现在可用于共聚焦显微镜（图4）。</li>
	<li>使用解剖显微镜观察SV组织，以确保其平放且不靠近任何气泡。如果SV组织的方向不正确，用户可以尝试推出气泡或小心地取出较小的玻璃盖玻片并重新定位SV。这必须轻轻地完成，以避免打破玻璃盖玻片。</li>
</ol>

用于单核测序或免疫染色的血管纹显微切割

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+irreversible+hearing+loss+is+caused+by+stria+vascularis+degeneration+in+an+Slc26a4-insufficient+mouse+model+of+large+vestibular+aqueduct+syndrome.">Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqueduct syndrome.</a> Neuroscience. 310, 188-197 (2015).</li><li>Gu, 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=Characterization+of+rare+spindle+and+root+cell+transcriptional+profiles+in+the+stria+vascularis+of+the+adult+mouse+cochlea.">Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea.</a> Scientific Reports. 10 (1), 18100 (2020).</li><li>Gow, 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=Deafness+in+claudin+11-null+mice+reveals+the+critical+contribution+of+basal+cell+tight+junctions+to+stria+vascularis+function.">Deafness in claudin 11-null mice reveals the critical contribution of basal cell tight junctions to stria vascularis function.</a> The Journal of Neuroscience. 24 (32), 7051-7062 (2004).</li><li>Chang, 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=Virally+mediated+Kcnq1+gene+replacement+therapy+in+the+immature+scala+media+restores+hearing+in+a+mouse+model+of+human+Jervell+and+Lange-Nielsen+deafness+syndrome.">Virally mediated Kcnq1 gene replacement therapy in the immature scala media restores hearing in a mouse model of human Jervell and Lange-Nielsen deafness syndrome.</a> EMBO Molecular Medicine. 7 (8), 1077-1086 (2015).</li><li>Faridi, 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=Mutational+and+phenotypic+spectra+of+KCNE1+deficiency+in+Jervell+and+Lange-Nielsen+Syndrome+and+Romano-Ward+Syndrome.">Mutational and phenotypic spectra of KCNE1 deficiency in Jervell and Lange-Nielsen Syndrome and Romano-Ward Syndrome.</a> Human Mutation. 40 (2), 162-176 (2019).</li><li>Wangemann, P., 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=Loss+of+KCNJ10+protein+expression+abolishes+endocochlear+potential+and+causes+deafness+in+Pendred+syndrome+mouse+model.">Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model.</a> BMC Medicine. 2, 30 (2004).</li><li>Kitajiri, S. -. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+analysis+of+single-cell+transcriptomic+data.">Cross-species analysis of single-cell transcriptomic data.</a> Frontiers in Cell and Developmental Biology. 7, 175 (2019).</li><li>Chen, G., Ning, B., Shi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-Seq+technologies+and+related+computational+data+analysis.">Single-cell RNA-Seq technologies and related computational data analysis.</a> Frontiers in Genetics. 10, 317 (2019).</li><li>Longo, S. K., Guo, M. G., Ji, A. L., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+single-cell+and+spatial+transcriptomics+to+elucidate+intercellular+tissue+dynamics.">Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics.</a> Nature Reviews Genetics. 22 (10), 627-644 (2021).</li><li>Kim, N., Kang, H., Jo, A., Yoo, S. A., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+single-nucleus+RNA+sequencing+in+different+cell+types+and+tissues.">Perspectives on single-nucleus RNA sequencing in different cell types and tissues.</a> Journal of Pathology and Translational Medicine. 57 (1), 52-59 (2023).</li><li>Grindberg, R. 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作者没有什么可透露的。

在单细胞测序出现之前，许多研究人员使用批量组织分析，这只能分析跨细胞平均的转录组。特别是，单细胞和sNuc-Seq使得分离单细胞或单核的转录组成为可能，分别32。在这种情况下，可以鉴定边缘细胞、中间细胞和基底细胞以及梭形细胞的单核转录组30。这使得能够研究SV细胞类型之间的转录异质性，并可用于将来研究这些细胞类型对SV功能的贡献，包括EP的?...

耳蜗由三个充满液体的腔室组成，即前庭鳞片、中鳞片和鼓鳞片。前庭鳞片和鼓鳞片均含有外淋巴液，其具有高浓度的钠（138 mM）和低浓度的钾（6.8 mM）1。Scala介质含有内淋巴液，其具有高浓度的钾（154mM）和低浓度的钠（0.91mM）1,2,3。这种离子浓度的差异可以称为耳蜗内电位（EP），主要是由钾离子沿着耳蜗侧壁通过血管纹（SV）中的各种离子通道和间隙连接产生的4,5,6,7,8,9,10,11 .SV是一种异质的，高度血管化的组织，排列在耳蜗侧壁的内侧，包含三种主要细胞类型：边缘细胞，中间细胞和基底细胞12（图1）。
边缘细胞通过紧密连接连接，形成SV的最内侧表面。顶膜面向斯卡拉介质的内淋巴，并通过各种通道（包括KCNE1 / KCNQ1，SLC12A2和Na + - K + - ATP酶（NKA）5,10,13,14）促进钾离子转运到内淋巴中。中间细胞是位于边缘细胞和基底细胞之间的色素细胞，并使用KCNJ10（Kir 4.1）15,16促进钾通过SV运输。基底细胞靠近耳蜗侧壁，与螺旋韧带的纤维细胞密切相关，以促进外淋巴12的钾循环。SV的病理学与许多耳科疾病有关17,18。在主要SV细胞类型（如Kcnq1，Kcne1，Kcnj10和Cldn11）中表达的基因突变可导致耳聋和SV功能障碍，包括EP19,20,21,22,23的丢失。除了三种主要细胞类型外，SV中还有其他研究较少的细胞类型，例如梭形细胞22，根细胞12,24，巨噬细胞25，周细胞26和内皮细胞27，其作用不完全确定，涉及离子稳态和EP 28的产生。
与批量RNA测序相比，单核RNA测序（sNuc-Seq）提供有关细胞异质性的信息，而不是一组细胞29的mRNA平均值，并且在研究异质SV30时特别有用。例如，sNuc-Seq已经产生了转录分析，表明纺锤体和根细胞可能在EP生成，听力损失和梅尼埃病中发挥作用18。各种SV细胞类型的进一步转录表征可以为我们提供有关SV相关听力波动和听力损失的不同机制和亚型病理生理学的宝贵信息。这些精细的内耳结构的收获对于最佳组织分析至关重要。
在本研究中，描述了从成年小鼠耳蜗中获取和分离血管纹以进行sNuc-Seq或免疫染色的显微解剖方法。需要解剖成年小鼠SV以了解各种SV细胞类型并进一步表征它们在听力中的作用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >10-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50010-01</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >18 x 18 mm cover glass</td>
			<td >Fisher Scientific</td>
			<td >12-541A</td>
			<td >Cover slip to mount SV</td>
		</tr>
		<tr >
			<td >30-&#181;m filter (Polyethylenterephthalat)</td>
			<td >PluriSelect</td>
			<td >#43-50030-03</td>
			<td >Filter tissue during sNuc-Seq</td>
		</tr>
		<tr >
			<td >75 x 25 mm Superfrost Plus/Colorforst Plus Microslide</td>
			<td >Daigger</td>
			<td >EF15978Z</td>
			<td >Microslide to mount SV on</td>
		</tr>
		<tr >
			<td >C57BL/6J Mice</td>
			<td >The Jackson Laboratory</td>
			<td >RRID: IMSR_JAX:000664</td>
			<td >General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >Cell Counter</td>
			<td >Logos Biosystems</td>
			<td >L20001</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Chalizon curette 5&#39;&#39;, size 3 2.5 mm</td>
			<td >Biomedical Research Instruments</td>
			<td >15-1020</td>
			<td >Used to transfer SV</td>
		</tr>
		<tr >
			<td >Chromium Next GEM single Cell 3&#39; GEM Kit v3.1</td>
			<td >Chromium</td>
			<td >PN-1000141</td>
			<td >Generates single cell 3&#39; gene expression libraries</td>
		</tr>
		<tr >
			<td >Clear nail polish</td>
			<td >Fisher Scientific</td>
			<td >NC1849418</td>
			<td >Used for sealing SV mount</td>
		</tr>
		<tr >
			<td >Corning Falcon Standard Tissue Culture Dishes, 24 well</td>
			<td >Corning</td>
			<td >08-772B</td>
			<td >Culture dish used to hold specimen during dissection</td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Invitrogen</td>
			<td >D1306, RRID: AB_2629482</td>
			<td >Stain used for nucleus labeling</td>
		</tr>
		<tr >
			<td >Dounce homogenizer</td>
			<td >Sigma-Aldrich</td>
			<td >D8938</td>
			<td >Used to homogenize tissue for sNuc-seq</td>
		</tr>
		<tr >
			<td >Dumont #5 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-30</td>
			<td >General forceps for dissection</td>
		</tr>
		<tr >
			<td >Dumont #55 Forceps</td>
			<td >Fine Science Tools</td>
			<td >11255-20</td>
			<td >Forceps with fine tip that makes SV manipulation easier</td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum</td>
			<td >ThermoFisher</td>
			<td >16000044</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Glue stick</td>
			<td >Fisher Scientific</td>
			<td >NC0691392</td>
			<td >Used for mounting SV</td>
		</tr>
		<tr >
			<td >GS-IB4 Antibody</td>
			<td >Molecular Probes</td>
			<td >I21411, RRID: AB-2314662</td>
			<td >Antibody used for capillary labeling</td>
		</tr>
		<tr >
			<td >KCNJ10-ZsGreen Mice</td>
			<td >n/a</td>
			<td >n/a</td>
			<td >Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.</td>
		</tr>
		<tr >
			<td >MgCl2</td>
			<td >ThermoFisher</td>
			<td >AM9530G</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Mounting reagent</td>
			<td >ThermoFisher</td>
			<td >#S36940</td>
			<td >Mounting reagent for SV</td>
		</tr>
		<tr >
			<td >Multiwell 24 well plate</td>
			<td >Corning</td>
			<td >#353047</td>
			<td >Plate used for immunostaining</td>
		</tr>
		<tr >
			<td >NaCl</td>
			<td >ThermoFisher</td>
			<td >AAJ216183</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nonidet P40</td>
			<td >Sigma-Aldrich</td>
			<td >9-16-45-9</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Nuclease free water</td>
			<td >ThermoFisher</td>
			<td >4387936</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Orbital shaker</td>
			<td >Silent Shake</td>
			<td >SYC-2102A</td>
			<td >Used for steps of immunostaining</td>
		</tr>
		<tr >
			<td >PBS</td>
			<td >ThermoFisher</td>
			<td >J61196.AP</td>
			<td >Used for steps of immunostaining and dissection</td>
		</tr>
		<tr >
			<td >RNA Later</td>
			<td >Invitrogen</td>
			<td >AM7021</td>
			<td >Used for preservation of SV for sNuc-Seq</td>
		</tr>
		<tr >
			<td >Scizzors</td>
			<td >Fine Science Tools</td>
			<td >14058-09</td>
			<td >Used for splitting mouse skull</td>
		</tr>
		<tr >
			<td >Tris-HCl</td>
			<td >Sigma-Aldrich</td>
			<td >15506017</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Trypan blue stain</td>
			<td >Gibco</td>
			<td >15250061</td>
			<td >Used for cell counting</td>
		</tr>
		<tr >
			<td >Tween20</td>
			<td >ThermoFisher</td>
			<td >AAJ20605AP&#160;</td>
			<td >Used for steps of sNuc-Seq</td>
		</tr>
		<tr >
			<td >Zeiss STEMI SV 11 Apo stereomicroscope</td>
			<td >Zeiss</td>
			<td >n/a</td>
			<td >Microscope used for dissections</td>
		</tr>
	</tbody>
</table>

dissection of adult mouse stria vascularis for single-nucleus sequencing or immunostaining

由血管纹产生的内耳蜗电位对于维持有利于适当的毛细胞机械转导和最终听力的环境至关重要。血管纹的病变可导致听力下降。解剖成体血管纹可以集中捕获单核，随后进行单核测序和免疫染色。这些技术用于在单细胞水平上研究血管纹的病理生理学。 
单核测序可用于血管纹的转录分析。同时，免疫染色在识别特定细胞群方面仍然有用。这两种方法都需要适当的血管纹夹层作为先决条件，这可能被证明在技术上具有挑战性。

我们提出了一种分离用于sNuc-Seq或免疫染色的SV的方法。耳蜗相对于SV的相关解剖结构（图1）可以帮助用户更好地了解SV的组织和解剖方案的步骤。
相关视频详细介绍了从P30鼠标显微解剖SV的每个步骤， 图2显示了这种解剖和分离SV的关键步骤的快照。
sNuc-Seq可用于研究异质SV中各种细胞的转录谱。一种可视化方?...

Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

血管纹对于内耳蜗电位的产生至关重要。在这里，我们介绍了成年小鼠血管纹的解剖以进行单核测序或免疫染色。

解剖成年小鼠血管纹以进行单核测序或免疫染色

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

血管纹的功能是产生耳蜗内电位并调节耳蜗中的离子稳态。听觉发育和恢复计划旨在了解将功能失调的离子稳态与听力和稳定性障碍联系起来的潜在机制，这些疾病是发生波动或突然听力损失的疾病。我实验室的工作已经建立了血管纹的转录景观，并有助于我们理解它在听力中的重要性。我们利用这些数据与知之甚少的听力和稳定障碍（包括梅尼埃病）进行了比较。该协议能够解剖和分离整个血管纹，以进行进一步的分子研究，包括免疫组织化学。鉴于血管纹细胞类型彼此错综复杂地交织在一起，核分离和单核RNA测序的使用促进了我们对所涉及的不同转录机制的理解。

dissection-adult-mouse-stria-vascularis-for-single-nucleus-sequencing

Research

JoVE Journal

Biology

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

2.2K 次观看.  National Institutes of Health. 血管纹的功能是产生耳蜗内电位并调节耳蜗中的离子稳态。听觉发育和恢复计划旨在了解将功能失调的离子稳态与听力和稳定性障碍联系起来的潜在机制，这些疾病是发生波动或突然听力损失的疾病。我实验室的工作已经建立了血管纹的转录景观，并有助于我们理解它在听力中的重要性。我们利用这些数据与知之甚少的听力和稳定障碍（包括梅尼埃病）进行了比较。该?...

视频: 作者聚焦： 用于单核测序或免疫染色的成年小鼠血管纹解剖  

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

从特定的大脑区域的RNA或蛋白质提取大鼠脑微解剖

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

科研

生物学

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

成年斑马鱼肾脏的解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

从成年小鼠肾脏中分离细胞核以进行单核RNA测序

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and the Plasmodium parasites that cause malaria, accumulate in the secretory cavities of SG cells. Here, they are poised for transmission to their vertebrate hosts during a subsequent blood meal. As adult glands form as an elaboration of larval SG duct bud remnants that persist beyond early pupal SG histolysis, the larval SG is an ideal target for interventions that limit disease transmission. Understanding larval SG development can help develop a better understanding of its morphology and functional adaptations and aid in the assessment of new interventions that target this organ. This video protocol demonstrates an efficient technique for isolating, fixing, and staining larval SGs from Anopheles gambiae mosquitoes. Glands dissected from larvae in a 25% ethanol solution are fixed in a methanol-glacial acetic acid mixture, followed by a cold acetone wash. After a few rinses in phosphate-buffered saline (PBS), SGs can be stained with a broad array of marker dyes and/or antisera against SG-expressed proteins. This method for larval SG isolation could also be used to collect tissue for in situ hybridization analysis, other transcriptomic applications, and proteomic studies.

Dissection and Immunostaining of Larval Salivary Glands from Anopheles gambiae Mosquitoes

The adult mosquito salivary gland (SG) is required for the transmission of all mosquito-borne pathogens to their human hosts, including viruses and parasites. This video demonstrates efficient isolation of the SGs from the larval (L4) stage Anopheles gambiae mosquitoes and preparation of the L4 SGs for further analysis.

冈 比亚按蚊 幼虫唾液腺的解剖和免疫染色

Mosquito salivary glands (SGs) are a requisite gateway organ for the transmission of insect-borne pathogens. Disease-causing agents, including viruses and ...

从冷冻哺乳动物组织中分离和分析单个细胞核

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.