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。

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

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  

解剖成年小鼠血管纹以进行单核测序或免疫染色

The stria vascularis functions to generate the endocochlear potential and regulate ion homeostasis in the cochlea. The auditory development and restoration program seeks to understand the underlying mechanisms connecting dysfunctional ion homeostasis with hearing and stability disorders, which are diseases where either fluctuation in or sudden hearing loss occurs. Work in my lab has established the transcriptional landscape of the stria vascularis and has contributed to our understanding of its importance in hearing.We have used these data to draw parallels to poorly-understood hearing and stability disorders, including Meniere's disease. This protocol enables the dissection and isolation of whole-mount stria vascularis for further molecular studies including immunohistochemistry. Given that stria vascularis cell types are intricately intertwined with one another, the use of nuclear isolation and single-nucleus RNA sequencing has facilitated our understanding of the distinct transcriptional machinery involved.

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

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。

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

1.3K 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  

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

科研

生物学

Mouse Inner Ear Extraction and Stria Vascularis Dissection

To begin, identify the inner ear within the temporal bone of a euthanized mouse. Scrape away the cranial nerves using number 55 forceps. Then use number 55 forceps to dissect out the bulla and capsule, detaching them from surrounding bone.Remove any remaining soft tissue from the inner ear surface. For SV dissection, use number 55 forceps to hold the specimen by the vestibular portion with the cochlear facing up. Pierce through the cochlear bone at the apex using the same forceps.Scrape along the apical turn, applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall. Using number 55 forceps remove cochlear bone pieces from the apical and middle turns.Gently pushing down the lateral wall, pry and remove the bone wall pieces towards the middle turn to expose the lateral wall of the apical and middle turns. Push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of the apical and middle turns from the spiral ganglion along the outer hair cell layer.Then remove pieces of spiral ganglion from inside the cochlear. Detach the cochlear from the vestibular portion of the temporal bone by inserting the forceps into the round and oval window and pushing down towards the vestibule to have better access to the lateral wall of the basal turn. After the cochlear is detached remove the vestibular portion of the inner ear.Prioritize preserving as much of the strayer as possible. Continue removing til the bone of the middle turn is detached from the SV.Next, remove pieces of the remaining cochlear bone covering the basal turn. This can be accomplished by pushing the lateral wall layer under the bone to detach it.Pry and gently pull the tissue to remove the farthest basal part of the lateral wall being careful not to damage the soft tissue below. With the basal part of the lateral wall detached, separate the lateral wall from the remaining cochlear by tracing the forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall until it reaches the apex.Then move the lateral wall to a fresh PBS. Lay the lateral wall flat revealing the SV as a darker layer of tissue on the internal side of the lateral wall. Gently pry the SV layer away from the lateral wall.Push aside the detached SV and advance the forceps along the lateral wall attempting to detach the SV as one long ribbon, trying not to squeeze the SV.Use a tissue scoop to collect the fragile tissue. Cochlear were dissected from adult mouse aged greater than or equal to postnatal day 30. Inner ear was extracted from the surrounding temporal bone.SV was exposed after the removal of bone covering the middle turn. The SV was fully separated from the lateral wall after dissection.

小鼠内耳提取和血管纹夹除术

Preparation of Stria Vascularis Single-Nucleus Suspension

Homogenize the dissected stria vascularis tissue from mouse ear in a two-milliliter Dounce homogenizer, with 10 to 20 strokes on ice. Lyse the tissue on ice for 25 minutes. Then, filter the tissue through a 30-micron filter and spin the filtrate at 500G for five minutes at four degrees Celsius.Remove the supernatant and resuspend the cell pellet in one milliliter of nuclei wash and resuspension buffer. Next, filter the cells through a 10-micron filter and centrifuge at 500G for five minutes at four degrees Celsius. Remove the supernatant and resuspend in 50 microliters of nuclei wash and resuspension buffer.After Trypan Blue staining, dilute five microliters of the cell suspension into 50 microliters of 1X PBS and count the cells. To prepare single nuclei captures, load the sample with the desired nuclear density onto the chip.

血管纹单核悬浮液的制备

Immunostaining of Mouse Stria Vascularis and Tissue Mounting

Prepare for immunostaining by dissecting the stria vascularis from the mouse and add the tissue to a 24-well plate containing 200 microliters of 4%paraformaldehyde in 1X PBS. Incubate the tissue for 20 minutes at room temperature. perform two short washes of 1X PBS on an orbital shaker at low RPM.Remove the PBS, then perform permeabilization and blocking for a minimum of one hour at room temperature in 300 microliters of PBST solution. Next, stain the tissue with the primary antibody overnight at four degrees Celsius. Then add a secondary antibody and incubate for two hours at room temperature on an orbital shaker at low RPM.Cover the plate to protect the fluorescently tagged secondary antibody from light. Next, prepare a larger microslide by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 by 18 millimeter glass cover slip. Place one drop of mounting reagent between the glue streaks.Using number 55 forceps, gab as little tissue as possible on one end of the SV, and transfer it from PBS to the mounting reagent. Place one end of the cover slip on the slide where one streak of glue was placed. Then gently release the cover slip to avoid creating air bubbles.Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount. Label the specimen on the glass cover slip, away from the visualization field. Visualize the SV tissue under a dissection microscope to ensure it is lying flat and not near any air bubbles.If the SV tissue is not oriented correctly, try to push out air bubbles or carefully remove the smaller glass cover slip and reposition the SV.A whole mount of SV from P30 mouse was prepared and confocal imaging was performed. ZsGreen expression was observed in the intermediate cells, GSIB4 labeled endothelial cells, and DAPI labeled the nuclei.

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

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