作者要感谢哈特维格·维博尔德的出色技术援助，以及汉堡-埃彭多夫大学医学中心的UKE显微镜成像设施（Umif）提供显微镜和支持。这项研究由DZHK（德国心血管研究中心）[FKZ 81Z4710141]资助。

所有涉及动物的程序都得到汉堡州动物护理和使用委员会（ORG870，959）和北莱茵-威斯特法伦州自然、环境和消费者保护局（LANUV，07/11）的批准，并符合国家卫生研究院的实验室动物护理和使用指南（2011年）。研究使用男性和女性（10-24周）C57BL/6小鼠（库存号000664，杰克逊实验室）和小鼠同源性（db/db）或异质性（db/het：控制）为糖尿病自发突变（Leprdb：Bks.Cg-Dock7m+/+ 勒普db/J，股票号000642，杰克逊实验室）。作者已经使用手头的协议，对60周以上的小鼠没有变化。
1. 木质刺结的定位和解剖
注：尽管描述和图纸大多在较大的物种中可用，但一些出版物以前曾分别使用解剖方法和荧光记者线描述SG在24 只大鼠和25 只小鼠中的位置。
<ol><li>准备50mL的冰冷（3-4°C）肝素化（20单位/mL，见 材料表）磷酸盐缓冲盐水（PBS）。在室温下对 SG 进行解剖 （RT）。</li>
	<li>根据机构和地方指南，通过吸入3%-5%异黄素对鼠标进行深度麻醉。通过失去踏板戒断反射来验证足够的麻醉。斩首小鼠或进行颈椎错位。 注：不正确的颈椎错位可能导致脊椎破裂和胸血管损伤，导致出血，从而阻碍交感链的准备或切除，使SG处于不正确的位置。因此，让有经验的人员在深度消沉中进行颈椎错位或斩首动物是至关重要的。</li>
	<li>用乙醇喷洒皮肤，用梅奥剪刀和窄图案钳子沿着前轴线打开胸腔。切开隔膜，取出肋骨的前部。</li>
	<li>使用伦敦钳子将主动脉和静脉卡瓦抓住隔膜上方，并用斯特拉比斯谟剪刀切割靠近下脊柱的所有血管和结缔组织，从而去除心肺包。</li>
	<li>使用塑料一次性移液器用肝素化 PBS 彻底冲洗胸腔，直到去除所有血液痕迹。</li>
	<li>将躯干置于立体显微镜制备双筒望远镜下，确保胸腔内具有外部光源的良好照明。</li>
	<li>找到第一根肋骨和朗格斯科利肌肉。 注：SG位于双边位置，平行于脊柱在第一根肋骨和脊柱之间的分支，在一个凹槽横向到隆格斯科利肌肉24。SG 的平侧位于朗格斯科利肌肉附近。根据准备情况，交感链的某些部分可能已经可见为与脊柱平行的白色斜纤维。这些可追溯到 SG。</li>
	<li>轻轻地使用杜蒙#5/45钳子的尖端，将结缔组织横向暴露在长腹肌上。</li>
	<li>将钳子转动 180°，然后使用平侧抓住 SG，并以最小的压力将其拉出。</li>
	<li>重复第二个 SG。</li>
	<li>将两个 SG 放在装满冷 PBS 的盘子（直径 6 厘米）中，并以适当的放大倍数进行检查。如有必要，去除多余的血管、脂肪组织和较大的神经。 注：自主结肠被一个结缔组织胶囊包围，由胶原纤维和成纤维细胞26，27组成。这些胶囊的渗透性似乎因物种而异，不同种类的黑帮26岁和28岁。使用 Dumont #5/45 钳子以及如有必要，使用弹簧剪刀去除尽可能多的结缔组织。</li>
	<li>根据实验目标，继续执行本协议的第 2、3 或 5 节。</li>
</ol>2. 全安装免疫造血化学协议
注：此协议是根据心脏整个安装染色4，29改编而成的。在 96 井板的一口井中为每个 SG 执行孵化步骤，并将 100 μL（用于含抗体解决方案）到 200 μL（用于所有其他解决方案）的解决方案进行孵化，以确保完全覆盖。定期用双筒望远镜检查 SG 的覆盖范围并正确浸入。手动取出液体时，配有 200 μL 移液器，在 200 μL 尖端上再加一个 10 μL 尖端。这将防止移液器尖端中 SG 的吸入。使用新制备的溶液和无菌液体来防止细菌生长。
<ol><li>修复 Sg 的组织学 2 小时在 Rt 在 4% 无甲醇副甲醛 （Pfa） / Pbs 。</li>
	<li>尽快处理 SG，但此时 PBS 中的钠酸钠含量为 0.02%（w/v），可在 PBS 的 4-6 °C 下存储 2-4 周。</li>
	<li>准备苏丹黑股解决方案（1%苏丹黑色w/v在100%乙醇），以减少自动荧光和改善信号到背景比30。在 RT 的磁搅拌器上溶解 2-3 小时。 注：使用库存溶液最多 6-8 周，在出现沉降时提前丢弃。</li>
	<li>准备苏丹黑色工作解决方案，通过离心的库存溶液30分钟全速（13，000 x g）去除碎片和稀释库存70%乙醇到最终浓度0.25%苏丹黑色。</li>
	<li>用登特的漂白剂治疗SG，以改善抗体渗透31。通过混合甲醇 （MeOH）、过氧化氢溶液 30% （w/w） 在 H2O 中和二甲基硫氧化物 （DMSO） 的比例为 4：1：1，新鲜准备登特的漂白剂。每 SG 添加 200 μL，并将板放置在轨道摇床上 1 小时 RT。</li>
	<li>在轨道摇床上孵育 10 分钟，进行降压 MeOH 系列以进行补液：100% MeOH、75% MeOH/PBS、50% MeOH/PBS、25% MeOH/PBS。</li>
	<li>在 RT 的 PBS/1% Triton-X-100 中，通过两次孵育 SG 进行 60 分钟，从而执行渗透。</li>
	<li>从 SG 中删除渗透解决方案，并添加苏丹黑色工作解决方案。在轨道摇床的 RT 上孵育 2 小时。</li>
	<li>同时，通过在 PBS 中加入 5% 受体级牛血清白蛋白 （BSA） 和 0.1% Triton-X-100（15 mL 容器），并让它在滚筒摇床上溶解约 5-10 分钟，从而准备阻塞溶液。通过预褶皱纸过滤器去除碎屑。</li>
	<li>通过倾斜板和小心地从直立侧管道去除苏丹黑色非常小心。 注：不可能看到苏丹的SG是黑色的。使用强光源并缓慢工作。从这一步开始，SG 被染黑，提高了可见度。如果此时在 SG 周围看到任何其他结缔组织，请使用 Dumont #5/45 钳子和弹簧剪刀将其取出。</li>
	<li>加入 200 μL 的 PBS/0.1% 特里顿-X-100 （PBS-T），在轨道摇床的 RT 上洗 5 分钟。</li>
	<li>用移液器吸气，然后重复 2 次，以去除 PBS-T。</li>
	<li>删除 PBS;加入 200 μL 的阻塞溶液，并在轨道摇床上在 4 °C 的夜间孵育。</li>
	<li>第二天，通过在阻塞溶液中添加主要抗体来准备解决方案。从既定协议中调整抗体浓度。 注：包括一个SG作为抗体控制没有原发性抗体（孵化与抗原预吸收抗体或IgG，如果可用，或阻塞缓冲）。</li>
	<li>在轨道摇床上执行 36-48 h 的初级抗体孵化，在 4 °C 下进行。对于细胞大小测量和交感神经元的染色，请使用抗酪氨酸羟基酶的抗体（请参阅抗体建议 材料表 ）。 注意：将 96 井板放在湿室（例如，内衬 ddH2O 湿纸巾的塑料盒），以防止此时蒸发。</li>
	<li>小心地去除抗体溶液，并添加 200 μL 的 PBS-T。将板放在轨道摇床上30分钟。</li>
	<li>移除 PBS-T 并重复洗涤步骤 5 次。</li>
	<li>使用前全速（13，000 x g）离心荧光亚历克萨标签的二级抗体，准备二次抗体工作解决方案1分钟。根据阻塞溶液中的主要抗体 1：500 稀释适当的二级抗体，如果需要核染色，则添加到 SG. 添加 1 μg/mL 的双苯酰胺 H33342 三氯化氢（霍奇斯特染色）。在轨道摇床的 4 °C 下孵育 12-24 小时。</li>
	<li>小心地取下抗体溶液，并添加 200 μL 的 PBS-T。将板放在轨道摇床上30分钟。</li>
	<li>移除 PBS-T 并重复洗涤步骤 5 次。对于最后一步，使用没有特里顿的 PBS。</li>
	<li>要嵌入，请将 50-100 μL 的荧光安装介质（参见 材料表）涂抹在玻璃滑梯上，并放置在准备的双筒望远镜下。使用 Dumont #5/45 钳子从 96 井板中取出 SG，并通过将一端浸入滤纸（例如 Whatman 干燥垫）中去除多余的液体，并将其放在掉落处。</li>
	<li>使用杜蒙#5/45 钳以适当的放大镜校正定位。</li>
	<li>轻轻地将玻璃盖片（20 毫米 x 20 毫米）放在 SG 旁边，然后慢慢下降。 注意：使用过多的安装介质会导致 SG 运动或神经缠绕。如果发生这种情况，请快速取下盖唇并重复步骤 2.9-2.21。</li>
	<li>让滑梯在 RT 的黑暗中干燥过夜。在 4 °C 下，至少可以储存 4-6 周。</li>
</ol>3. 全安装就地杂交
注：SG的全安装原位混合体由corti32 器官和商业RNA荧光原位协议（见 材料表）改编而成。从供应商那里获得感兴趣的基因、缓冲和解决方案的探针。所有孵化步骤均在 RT 执行，如果不提及，则不进行。使用无菌 PBS。如果有兴趣在一口井中染色多个 SG，请使用至少 150 μL 的缓冲器和解决方案。
<ol><li>执行步骤 1.1-1.13 中描述的 SG 解剖。</li>
	<li>固定 SG 1 h 在 200 μL 的 4% 无 MEOH PFA/PBS 在一口井的 96 井板放置在轨道摇床。</li>
	<li>在轨道摇床上以 0.1% Tween-20/PBS 为 30 分钟清洗 SG 三次。</li>
	<li>脱水 SG 在 MeOH/PBS 系列中，通过随后在 50% MeOH/PBS、70% MeOH/PBS 和 100% MeOH 中孵育，每个在轨道摇床上 10 分钟。</li>
	<li>将 SG 存储在 -20 °C 的 100% MeOH 过夜。</li>
	<li>第二天，将孵化器预热至40°C。 使用温度计检查温度。</li>
	<li>在反向 MeOH/PBS 系列（100% MeOH、70% MeOH/PBS、50% MeOH/PBS）中补充水合 SG，每次 10 分钟。</li>
	<li>在 PBS 中，每次清洗 SG 三次，每次 5 分钟。</li>
	<li>同时，在 40 °C 的潜伏期 10 分钟开始预热探测器，然后冷却 10 分钟。</li>
	<li>在 200 μL 的蛋白酶 III 中孵育 SG 15 分钟。</li>
	<li>可选：如果计划进行后续免疫荧光染色，则根据本协议第 2.3、2.4 和 2.8 节执行苏丹黑色处理以淬火自动荧光。</li>
	<li>在轨道摇床上以 0.1% Tween-20/PBS 3x 的 200 μL 清洗 SG，每次 5 分钟。</li>
	<li>可选：如果需要与多个探头共染污，则稀释通道-2 （50 倍） 和第 3 频道探头 （50 倍） 在第 1 频道探头 （1x）中。</li>
	<li>用100微升的探针覆盖SG，寻找感兴趣的基因，并在40°C下在夜间孵育，并轻微激动。将 96 井板放置在 40 °C 的湿室中，用于所有孵化步骤。 注：包括一个SG作为负控制，使用对细菌基因（如二氢二聚二甲酸酯还原酶 ，Dapb）的探针，以检查在以后的步骤中放大试剂的非特异性结合。</li>
	<li>在提供洗涤缓冲器中清洗 SG，每次在轨道摇床上清洗 15 分钟。</li>
	<li>预热 Amp1-3、HRP-C1 和 HRP 拦截器到 RT。如果需要与通道-2和/或通道-2探头共同染色，则需要预热 HRP-C2 和 HRP-C3。</li>
	<li>在轨道摇床上以 4% PFA/PBS 在 RT 中重新修复 SG 10 分钟。</li>
	<li>在 Rt 的轨道摇床上以 200 μL 的供应洗涤缓冲器 3 倍清洗 Sg， 每次 5 分钟。</li>
	<li>为了放大，在轨道摇床上用 100 μL 的 Amp1 孵育 SG，在 40 °C 下进行 35 分钟。</li>
	<li>小心地取出任何液体，在轨道摇床的 RT 上用 200 μL 的供应洗涤缓冲器 3x 清洗 SG，每次 5 分钟。</li>
	<li>在轨道摇床上用 100 μL 的 Amp2 孵化 SG，在 40 °C 下进行 35 分钟。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上用 100 μL 的 Amp3 孵化 SG 20 分钟，在 40 °C 下。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上以 100 μL 的多路复用 FL v2 HRP-C1 孵育 SG 20 分钟，在 40 °C 处使用。</li>
	<li>重复步骤 3.18。</li>
	<li>准备蛋白石结合的二级抗体 1：1，000 在 200 μL 提供的 TSA 缓冲和孵育 SG 35 分钟在 40 °C 的轨道摇床。在孵化过程中和从这一步开始时，要防止光线照射。</li>
	<li>重复步骤 3.18。</li>
	<li>在轨道摇床上以 100 μL 的多路复用 FL v2 HRP 阻滞器在 40 °C 处孵育 SG 15 分钟。</li>
	<li>重复步骤 3.18。</li>
	<li>可选：用于与通道-2探头共同染色，重复步骤 3.25-3.30 与提供的多路复用 FL v2 HRP-C2。</li>
	<li>可选：用于与通道-3探头共同染色，重复步骤 3.25-3.30 与提供的多路复用 FL v2 HRP-C3。</li>
	<li>如果随后发生免疫荧光染色，执行本协议的第 2.13-2.25 步。</li>
	<li>在轨道摇床上以 1% BSA/PBS 孵育 30 分钟。</li>
	<li>如果需要核染色，在 1 μg/mL 的双苯甲酰胺 H33342 三氯化氢 （霍奇斯特染色） 中孵育 SG 30 分钟，如果需要核染色，并/或在轨道摇床上添加亚历克萨耦合小麦胚芽凝胶素 （WGA， 1：500）。</li>
	<li>重复步骤 3.18。</li>
	<li>按步骤 2.19-2.22 中描述的嵌入。</li>
</ol>4. 木质结肠的成像和分析
<ol><li>在本地成像设备中对嵌入式 SG 进行共聚焦显微镜检查。</li>
	<li>如果需要细胞大小测量，图像SG染色酪氨酸羟基酶（见 材料表）在200倍放大，并采取4-6随机图像从每个SG。</li>
	<li>使用 ImageJ33 软件分析图像以估计单元格大小（例如，使用笔表，参见 材料表）。使用 免费手选择，圈每个单元格，然后单击 分析|测量 获取细胞区域。小心只包括位于 SG 内的完整、完全可见的细胞。</li>
	<li>使用此方法，执行每 SG 约 100 个细胞的测量。</li>
	<li>如果你想比较来自不同老鼠的 SG，请让失明的调查员执行步骤 4.2-4.3。使用统计软件中的频率分布来可视化第 34组之间的大小差异。</li>
</ol>5. 穆林·斯特拉特结帮的分子分析
注意：根据您的实验设计，包括控制。这可能是具有不同基因型和疾病背景和/或其他自主结节的SG，如交感优越的宫颈结节（位于颈部区域，见齐格勒等人的详细描述）或寄生性结节（如颅内结节，见君根等人）。
<ol><li>准备一个2mL管与500微升的酚/瓜尼丁硫氰酸酯溶液（例如，Qiazol）每只动物，并有液氮准备防震霜或考虑商业解决方案，以保护RNA（可选，见材料表）36。快速进行RNA隔离。</li>
	<li>执行步骤 1.1-1.13 中描述的 SG 解剖。</li>
	<li>立即将两个 SG 直接浸入一个管中，用酚/瓜尼丁硫氰酸酯溶液和冲击霜管浸入液氮中。</li>
	<li>在 -80 °C 下存放，直到进一步处理。</li>
	<li>对于组织裂解，让带有 SG 解冻的管子解冻，直到苯酚/瓜尼丁硫氰酸酯溶液液液化，并添加两个 7 mm 不锈钢珠。在 4 °C 的干冰和离心机上冷却组织同质化器（球或搅拌机，例如组织 Lyser II）的金属部件。</li>
	<li>离心管在 500 x g 1 分钟在 4 °C，使 SG 在管的底部。</li>
	<li>将管子放入组织莱瑟的金属部件中，在 20 Hz 处将 1 分钟置于莱塞和莱塞中 1 分钟。</li>
	<li>重复步骤 5.6 和 5.7 最多 5 次，直到无法检测到完整的组织。</li>
	<li>将液体转移到新鲜的 1.5 mL 管中。</li>
	<li>根据制造商的说明，使用基于柱的RNA隔离套件（例如 miRNeasy 迷你套件）执行 RNA 隔离。</li>
	<li>Elute RNA 在 20 μL 的无 RNase 水中，使用光谱光度计测量浓度。 注：为了排除纯RNA与基因组DNA的污染，我们建议用基因组引物和1微升RNA作为模板进行聚合酶链式反应，而不是减去反向转录酶控制。这将节省大量的RNA。如果RNA被污染，请使用外在内子边界引物或内侧引物进行后续定量实时聚合酶链反应。</li>
	<li>使用 250 ng SG RNA 执行 cDNA 合成并使用已建立的协议。在这里，根据制造商的说明使用大容量 cDNA 反向转录套件。</li>
	<li>稀释到 cDNA 的 2.5 ng/μL 的最终浓度，并根据既定协议与适当的探头进行定量实时聚合酶链式反应。在这里，塔克曼检测（见 材料表）使用10 ng cDNA每反应进行。 注：对每个基因执行无模板控制，以排除误报结果。</li>
	<li>使你感兴趣的基因在家养基因上的基因表达正常化（例如 ，Cdkn1b），以比较不同组的SG之间的相对基因表达。</li>
</ol>

作者没有什么可透露的。

对VA发病前交感神经系统神经元和胶质细胞的细胞和分子过程的理解是高度关注的，因为心脏骤停仍然是全世界最常见的死因。因此，在当前的手稿中，我们提供了一系列基本的方法来识别该网络中的穆林 SG - 喃喃元素 ， 并随后对 RNA、蛋白质和细胞水平进行分析。
Murine SG的一个挑战是它的大小和有限的细胞数量39。因此，不同的动物模型?...

心脏自主神经系统是一种受严格控制的交感、寄生和感官成分的平衡，使心脏能够适应环境变化，并具有适当的生理反应1，2。这种平衡的干扰，例如，同情活动的增加，已被确定为发病和维持心室心律失常（VA）3，4的关键驱动因素。因此，自主调制，通过药理学减少与β阻滞剂的交感活性，一直是治疗VA患者几十年的基石5，6。但是，尽管药理学和导管为基础的干预，相关数量的患者仍然患有复发性VA7。
对心脏的交感输入主要通过交感链的双星形结构——神经元细胞体进行中介，这种结构通过无数的胸内神经从脑干传递信息到心脏8、9、10。受伤后从SG发芽的神经与VA和心脏骤死11，12有关，强调SG作为自主调制的目标13，14。通过局部麻醉剂的皮下注射或通过视频辅助胸腔镜检查15、16部分切除SG，可以暂时减少对心脏的交感输入。心脏交感衰竭为治疗难治VA的患者提供了一个选择，结果有希望的结果14，16，17。我们从这些患者的外植SG中了解到，神经元和神经化学改造、神经炎症和胶质活化是同情性重塑的标志，可能有助于或加重自主功能障碍18，19。尽管如此，这些神经元中潜在的细胞和分子过程至今仍然模糊不清，例如，神经元分化成胆碱表型20，21的作用。实验研究提出了治疗VA的新方法，例如，通过光遗传学22减少交感神经活动，但SG的深度表征仍然缺乏许多心脏病理与VA齐头并进。模仿这些病理学的小鼠模型可以研究神经元重塑，这可能先于心律失常12，23的发生。这些可以通过进一步的形态和功能分析来完成，以实现心脏和神经系统的自主特征。在本协议中，我们提供了一系列基本的方法，允许解剖和描述 Murine SG，以增进对 VA 的理解。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>TPP</td>
			<td>92097</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Adhesion Slides SuperFrost plus&#160; 25 x 75 x 1 mm</td>
			<td>R. Langenbrinck</td>
			<td>03-0060</td>
			<td>Microscopy</td>
		</tr>
		<tr>
			<td>Albumin bovine Fraction V receptor grade lyophil.</td>
			<td>Serva</td>
			<td>11924.03</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>bisBenzimide H33342 trihydrochloride (Hoechst)</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>B2261</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Chicken anti neurofilament</td>
			<td>EMD Millipore</td>
			<td>AB5539</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>D8418</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti chicken IgY Alexa 647&#160;</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>AP194SA6</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti goat IgG Alexa 568&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11057</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Donkey anti rabbit IgG Alexa 488&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21206</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Drying block 37-100 mm</td>
			<td>Whatman (Sigma Aldrich)</td>
			<td>WHA10310992&#160;</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma Aldrich</td>
			<td>E4009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Ethanol 99 % denatured with MEK, IPA and Bitrex (min. 99,8 %)</td>
			<td>Th.Geyer</td>
			<td>2212.5000</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Eukitt mounting medium</td>
			<td>AppliChem</td>
			<td>253681.0008</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Fluoromount-G + DAPI</td>
			<td>Southern Biotech</td>
			<td>0100-20</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Goat anti choline acetyltransferase</td>
			<td>EMD Millipore</td>
			<td>AP144P</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>H2O2 30% (w/w)</td>
			<td>Merck, KGA, Darmstadt, Germany</td>
			<td>H1009</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Heparin Sodium 25.000 UI / 5ml</td>
			<td>Rotexmedica</td>
			<td>PZN: 3862340</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>High-capacity cDNA reverse transctiption kit</td>
			<td>Life technologies</td>
			<td>&#160;4368813</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Isoflurane (Forene)</td>
			<td>Abbott Laboratories</td>
			<td>2594.00.00</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>Mayer&#39;s hemalum solution</td>
			<td>Merck</td>
			<td>1.09249.0500</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Microscope cover glasses 20x20 mm or smaller</td>
			<td>Marienfeld</td>
			<td>0101040</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>miRNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>217004</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>NanoDrop 2000c</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000C</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Opal 570 Reagent Pack</td>
			<td>Akoya Bioscience</td>
			<td>FP1488001KT</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aq. soln., methanol free&#160;</td>
			<td>Alfa Aesar</td>
			<td>43368</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pasteur pipettes, LDPE, unsterile, 3 ml, 154 mm</td>
			<td>Th.Geyer</td>
			<td>7691202</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline tablets</td>
			<td>Gibco</td>
			<td>18912-014</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Pinzette Dumont SS Forceps</td>
			<td>FineScienceTools</td>
			<td>11203-25</td>
			<td>Preparation SG</td>
		</tr>
		<tr>
			<td>QIAzol Lysis Reagent</td>
			<td>Qiagen&#160;</td>
			<td>79306</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Rabbit anti tyrosine hydroxylase</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Merck</td>
			<td>R0901-100ML</td>
			<td>RNA isolation (optional)</td>
		</tr>
		<tr>
			<td>RNAscope Multiplex Fluorescent Reagent Kit v2</td>
			<td>biotechne (ACD)</td>
			<td>323100</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-S100b-C2</td>
			<td>biotechne (ACD)</td>
			<td>431738-C2</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>RNAscope Probe-Mm-Tubb3</td>
			<td>biotechne (ACD)</td>
			<td>423391</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Stainless steel beads 7 mm&#160;</td>
			<td>Qiagen&#160;</td>
			<td>69990</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Sudan black B</td>
			<td>Roth</td>
			<td>0292.2</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Cdkn1b (Mm00438168_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Choline acetyltransferase (Mm01221880_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay MKi67 (Mm01278617_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay PTPCR (Mm01293577_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay S100b (Mm00485897_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan Gene Expression Assay Tyrosin Hydroxylase (Mm00447557_m1)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4331182</td>
			<td>Gene expression analysis</td>
		</tr>
		<tr>
			<td>TaqMan mastermix</td>
			<td>Applied biosystems</td>
			<td>4370074</td>
			<td>Gene Expression analysis&#160;</td>
		</tr>
		<tr>
			<td>Tissue Lyser II</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td>RNA isolation</td>
		</tr>
		<tr>
			<td>Triton X-100 10% solution</td>
			<td>Sigma-Aldrich</td>
			<td>93443-100ml</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416-100ML</td>
			<td>RNAscope</td>
		</tr>
		<tr>
			<td>Wacom bamboo pen</td>
			<td>Wacom</td>
			<td>CTL-460/K</td>
			<td>Cell size measurements</td>
		</tr>
		<tr>
			<td>Whatman prepleated qualitative filter paper, Grade 595 1/2</td>
			<td>Sigma-Aldrich</td>
			<td>WHA10311647</td>
			<td>Whole mount staining</td>
		</tr>
		<tr>
			<td>Wheat Germ Agglutinin, Alexa Fluor 633 Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>W21404</td>
			<td>RNAscope</td>
		</tr>
	</tbody>
</table>

location, dissection, and analysis of the murine stellate ganglion

自主神经系统是心脏电生理学的一大驱动力。特别是其同情分支的作用是心室心律失常（VA）病理生理学中正在进行的研究。交感链的双子星形结构（SG）   中的神经元   是交感基础设施的重要组成部分。SG 是治疗难治 VA 患者通过心脏交感衰减而公认的治疗目标。虽然在SG的神经元重塑和胶质激活已经描述了VA患者，潜在的潜在的细胞和分子过程，潜在的心律失常开始之前，只是不够了解，应该阐明，以改善自主调节。小鼠模型允许我们研究交感神经元改造，但对缺乏经验的研究者来说，识别穆林SG是具有挑战性的。因此，许多常见的心脏病缺乏对穆林SG的深入细胞和分子生物学研究。在这里，我们描述了解剖和研究成年小鼠SG的基本剧目，用于RNA水平分析（基因表达分析的RNA分离，原位杂交）、蛋白质水平（免疫荧光全安装染色）和细胞水平（基本形态、细胞大小测量）。我们提出潜在的解决方案，以克服制备技术中的挑战，以及如何通过淬火自流来改善染色。这允许通过既定标记对神经元和胶质细胞进行可视化，以确定细胞组成和重塑过程。这里介绍的方法允许SG的特征获得关于容易发生VA的小鼠自主功能障碍的进一步信息，并可以辅之以额外的技术来研究心脏自主神经系统的神经元和胶质成分。

图 1可视化如何识别和解剖 SG.图 1A显示了位置的示意图图，而图 1B在切除心肺包后将视图呈现到胸腔中。左和右长部的科利肌肉从 SG 和肋骨笼是方向的重要地标。解剖是沿着肌肉和第一根肋骨之间的虚线进行的。SG 和交感链变得可见为白色结构（图 1C）。图 1D显示了左长腹肌和第一根...

Watch this Scientific Journal Video about Location, Dissection, and Analysis of the Murine Stellate Ganglion at JoVE.com

Location, Dissection, and Analysis of the Murine Stellate Ganglion

心脏自主神经系统的病理生理变化，特别是其交感分支的病理生理变化，有助于心室心律失常的发病和维持。在本协议中，我们展示了如何描述粘膜结节，以增进对基础分子和细胞过程的理解。

穆林·斯特拉特·甘利翁的位置、解剖和分析

The autonomic nervous system is a substantial driver of cardiac electrophysiology. Especially&#160;the role of its sympathetic branch is an ongoing matter ...

location-dissection-and-analysis-of-the-murine-stellate-ganglion

Research

JoVE Journal

Medicine

9.5K 次观看.  EVK Düsseldorf, cNEP, cardiac Neuro- and Electrophysiology Research Consortium. 心脏自主神经系统的病理生理变化，特别是其交感分支的病理生理变化，有助于心室心律失常的发病和维持。在本协议中，我们展示了如何描述粘膜结节，以增进对基础分子和细胞过程的理解。

视频: Location, Dissection, and Analysis of the Murine Stellate Ganglion

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

小鼠胰岛的分离和包膜下肾移植的方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

科研

医学

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

人类蛋白质组分析玻璃体元素剖析

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have been developed to facilitate the study of the molecular and cellular pathophysiology of this disease. In this manuscript we describe specific techniques for the quantification and characterization of atherosclerotic lesions in the murine aortic sinus and ascending aorta. The advantage of this procedure is that it provides an accurate measurement of the cross-sectional area and total volume of the lesion, which can be used to compare atherosclerotic progression across different treatment groups. This is possible through the use of the valve leaflets as an anatomical landmark, together with careful adjustment of the sectioning angle. We also describe basic staining methods that can be used to begin to characterize atherosclerotic progression. These can be further modified to investigate antigens of specific interest to the researcher. The described techniques are generally applicable to a wide variety of existing and newly created dietary and genetically-induced models of atherogenesis.

We describe procedures to quantify and characterize atherosclerotic lesions in mouse models using precision sectioning of the aortic sinus and ascending aorta combined with histochemical and immunohistochemical analysis.

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝转移的临床前小鼠模型

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous studies have shown the results of such dissections, the detailed steps have not been described, precluding researchers outside the field from replicating their methods. Other studies harvest vitreous either through aspiration, which does not maintain the vitreous structure anatomy, or through partial dissection, which only isolates the vitreous core. The proposed method isolates the whole vitreous body, with the vitreous core and cortex intact, while maintaining vitreous anatomy and structural integrity. In this method, a full thickness scleral flap in an enucleated porcine eye is first created and through this, the choroid tissue can be separated from the sclera. The scleral flap is then expanded and the choroid is completely separated from the sclera. Finally the choroid-retina tissue is peeled off the vitreous to leave an isolated intact vitreous body. The proposed vitreous dissection technique can be used to study physical properties of the vitreous humor. In particular, this method has significance for experimental studies involving drug delivery, vitreo-retinal oxygen transport, and intraocular convection.

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes.

全玻璃体液清扫Vitreodynamic分析

The authors propose an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine eyes. While previous ...

Murine Prostate Micro-dissection and Surgical Castration

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has been used as a surrogate for discoveries in human prostate development and disease. Prostate micro-dissection allows consistent study of lobe-specific prostate anatomy, histology, and cellular characteristics in the absence of contamination of other tissues. Testosterone affects prostate development and disease. Androgen deprivation therapy is a common treatment for prostate cancer patients, but many prostate tumors become castration-resistant. Surgical castration of mouse models allows for the study of castration resistance and other facets of hormonal biology on the prostate. This procedure can be coupled with testosterone reintroduction, or hormonal regeneration of the prostate, a powerful method to study stem cell lineages in the prostate. Together, prostate micro-dissection and surgical castration opens up a multitude of opportunities for robust and consistent research of prostate development and disease. This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse.

This manuscript describes the protocols for prostate micro-dissection and surgical castration in the laboratory mouse. We also depict representative results produced by these protocols. Finally, we discuss the advantages and utilization of these protocols.

鼠前列腺微解剖和手术去势

Mouse models are used extensively to study prostate cancer and other diseases. The mouse is an excellent model with which to study the prostate and has ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

解剖的小鼠胰腺的组织学分析与代谢谱

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized &#945;4&#946;1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

We describe an in vitro assay to model chorioallantoic attachment, the first step in placenta formation. The protocol demonstrates the dissection and explant culture of murine allantoides on immobilized &#945;4&#946;1 integrin. Allantois attachment is evaluated microscopically at pre-determined time points.

小鼠尿的解剖和外植体培养对囊附着的作用分析

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. 
In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.

Here, we present the cyclic loading-induced intra-articular cartilage lesion model of the rat knee, generated by 60 cyclic compressions over 20 N, resulting in damage to the femoral condylar cartilage in rats.

一种生成周期负荷诱导的大鼠膝关节内软骨病变模型的非侵入性方法

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

使用吲哚菁绿荧光成像进行机器人左肝切除术治疗肝内复合胆囊肿

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...