我们感谢杰夫·塔克，埃丽卡·斯卡皮尼，和阿格奈什·贾诺沙齐他们用显微镜的帮助下，利贡·佩罗她的鼠标殖民地的管理，朱·切和迈克尔·山德森与组织切片机的帮助下，和迈克尔·费斯勒和德里克该隐的批判性阅读手稿。这项工作是由NIEHS，美国国立卫生研究院（ZIA ES102025-09），后者又由卫生和人类服务部赞助的校内分行资助。

本文中所描述的动物实验过程由NIEHS动物护理和使用委员会（IACUC）的批准。 1.肺制备<ol><li> 6和12周龄无特定病原体条件之间家鼠按照机构动物护理和使用委员会提供的指导原则。 注:成像小鼠可以是天真的，或经处理的，这取决于每个研究人员的具体利益。在这里，我们描述了在天真的小鼠和在用100μg卵清蛋白（OVA）和0.1微克的脂多糖（LPS）处理的小鼠中的免疫细胞定位，使用磷酸盐缓冲盐水（PBS）在50微升的总体积的车辆。到oropharyngeally灌输OVA / LPS进入气道，将动物用异氟烷麻醉，吸入并通过用橡胶带齿垂直悬挂。舌头轻轻地用钳子抓住并持有偏向一侧，防止吞咽和50＆＃181;保藏在口腔后部的OVA / LPS溶液的L，以先前描述的17。 </li><li>安乐死用戊巴比妥钠（100毫克/千克）的腹膜内注射小鼠，并钉住动物对于聚苯乙烯基。 </li><li>通过从腹部到颚的中间切开皮肤和腹膜打开用剪刀腹腔。用钳子或剪刀的钝边一边拉肠，和剪断下腔静脉从肺部排出血液程。穿刺用剪刀的尖端隔膜以允许所述左肋膨胀，小心不要切断肺部。 </li><li>清除唾液腺和通过抓住组织并手动从与钳子底层气管拉动远离其它组织从气管程。使使用细镊子或剪刀软骨厚带的前侧气管一小切口，小心不要一路砍虽然TRA谢。该切口将只是大到足以让一个20号针通过。 </li><li>滑动的1.5英寸，20号针上的聚乙烯管的截面，留下约1cm管超过针的端部。切管以大约45°的角度通过拉伸0.8毫升热水（40℃）2％低熔点琼脂糖向上穿过针斜面端部，附接针1mL注射器，并加载注射器。 </li><li>将管的一端插入气管的切口，并缓缓注入琼脂糖进入肺部。而无需移动针或注射器，用胶带将注射器的聚苯乙烯基础，以确保针头留在气管中。 注意:注射后，肺部将胸腔内较大和充气。右叶首先展开，然后左叶。如果只有一个叶扩大，针已经插入过深（过去支气管），而且有必要在继续之前把它拉出来咯。的这部分过程需要相对较快地完成，琼脂糖开始凝固之前。 </li><li>将鼠标，与基座和在气管仍插注射器，在冷室或冰箱中至少10分钟。如果有必要，动物可以在那里保存长达几个小时，即使通过视频显微镜在进行图像细胞迁移。 </li><li>一旦鼠标是冷的，用剪刀仔细切除琼脂糖充气肺，并将它们放置在一个3 cm培养皿用冰冷的PBS。在冰上保持。 </li><li>选择用于组织切片所需的波瓣（ 例如 ，右上叶）。确保肺（它连接到气管）的内饰部分是面朝下的菜。 注:所用的叶和活塞上的取向将决定血管和气管，这将是可见的大小。这里所描述的方位非常适合大型航空公司的可视化和实质。老鼠有一个大的左叶和右四个小瓣。对于以下介绍过敏原成通过口咽或鼻内抽吸肺PCLS成像，右上叶通常剖开，部分是因为它是一种方便的大小，而且还因为吸入剂在其内均匀地分散。然而，其他叶，尤其是左叶，也可以研究。当比较小鼠品系或治疗，它可以比较同叶是很重要的。 </li></ol> 2.肺切片注意:请用自动切片机，金属冷却块，柱塞和注射器金属片，根据制造商的说明。 <ol><li>放的所有目的的小滴，没有润凝胶强力胶到柱塞和散布胶水的圆周运动，同时注意不要让胶水接触金属注射器的侧面。如果胶水接触到注射器的两侧，将胶针筒和柱塞在一起。 </li><li>覆盖用胶水活塞后，立即轻轻抓住用与气管侧朝下钳子切叶，DAB它上的组织，以除去过量的液体，然后小心地将其放置在柱塞的顶部。剪掉延伸超出所述柱塞的边缘的任何额外的组织。 </li><li>向下移动柱塞，使得金属注射器的侧面向上移动，并通过组织，从而形成井在底部胶合，不超过几个厘米深肺。周围的金属注射器的底部胶带举行的地方这个位置。 </li><li>小心倾2％低熔点琼脂糖在40℃下到井中以便它只是覆盖波瓣的顶部。 </li><li>包围与冰冷的冷却块中的金属注射器和冷却叶和琼脂糖为1 - 2分钟。 注:周围组织中的琼脂糖的更短的时间冷却可以切片，这可能会导致切割不均匀PCLS期间导致琼脂糖的崩解。 </li><li>加载样品注射器到自动限幅器和填充用冰冷的PBS缓冲罐。对齐<html>步骤与试样注射器马达驱动，并从底部移除胶带片。转切换到快进（FF）的位置，直到所述步进电机驱动器刚好接触到柱塞的背面。 </li><li>对齐与样品注射器新的刀片。集合组织厚度，连续/单切片，振荡和速度在限幅器。例如，组织厚度:150微米，连续切削（续），振荡:9，和速度:3 - 4.按开始。通过以上的设置需要调整到特定的自动切片机规格。 </li><li>使用薄的抹刀或画笔，收集PCLS一次一个，因为它们落入缓冲罐和将它们放置在含有冰冷的PBS 24孔板中。 注:保持片，以保持实验之间的一致性是非常重要的。虽然每个肺是根据年龄，性别和重量不同，则10 个切片，例如，通常能产生类似大小的气道。 </li></ol>ove_title"&gt; 3。抗体染色<ol><li>设计基于对感兴趣的细胞类型的荧光标记抗体的面板中，考虑到潜在的荧光光谱重叠和显微镜的检测能力。 注意:作为一个例子，对于DC子集检测一个面板是如下:的CD11c /α-X整 - BrilliantViolet（BV）605（激发:405纳米，峰值发射:605纳米），CD324 / E-钙粘蛋白 - 的Alexa 488（AF488 ）（激发:488纳米，峰值发射:519纳米），CD88 / C5Ra1 - 藻红蛋白（PE）（激发:561纳米，峰值发射:578纳米），CD103 /α-ë整 - 别藻蓝蛋白（APC）（激发:633纳米，峰值发射:660纳米）。荧光光谱查看器工具（参阅材料清单）是设计的面板非常有用。 </li><li>使含有所需抗体的抗体混合物。 <ol><li>对于静态PCLS成像，添加800μL的染色缓冲液，100微升弗克·布洛克，50μL正常小鼠血清，和50μL正常大鼠血清到1.5mL microcentrifugation管1毫升的总体积。活细胞成像，使用含有10％FBS（的Leibovitz-10）代替染色缓冲液800μL的Leibovitz培养基（1×）。 </li><li>添加抗体来调节每种抗体的所期望的终浓度。转移抗体溶液到3cm的盘，并保持在黑暗中待用。 注:（ - 5微克/毫升通常1种）的最终浓度需要为每个单独的抗体进行优化。 </li></ol></li><li>选择与所述兴趣，如大气道或肺的周围的解剖区域中的PCLS，并在含有1种毫升抗体溶液中的3-cm培养皿放置。 </li><li>污点PCLS在黑暗中，在冰上，慢慢摇动30或60分钟。 </li><li>对于静态PCLS成像，进行到该部分4。对于活细胞成像，则继续执行第5节。 </li></ol> 4. PCLS的静态影像<ol><li>后用抗体PCLS的60分钟温育后，除去从培养皿抗体溶液和冲洗吨他用1个毫升冰冷的PBS切片两次。 </li><li>移液管加入50μLPBS涂到3厘米的圆形玻璃底培养皿中。使用刮刀或画笔，染色PCLS转移到滴，轻轻操纵切片，直到它是平坦的并且展开。用吸管取出PBS。切片应位于尽可能平坦。上述程序需要快速执行，以避免光漂白。 </li><li>放置室温下的安装介质1滴置于该片中，轻轻地滴在玻璃盖玻片到板的孔中。保持PCLS在黑暗中在4℃下最多成像前一个小时。 </li><li>当共焦显微镜下观察，使用20X物镜（参见材料列表 ）。使用"瓦片"工具中的"凸包"的设置来定位和标记组织的边缘。这将有助于减少瓷砖扫描的时间。 </li><li>而设置的z堆栈范围，验证在切片的多个区域，特别是沿着边缘，该组范围是合适的。 注:Z-范围将可能需要比组织本身的厚度更大，因为它是很难得到的组织奠定完全平坦。例如，具有150微米厚的PCLS中，z堆栈范围将可能需要被设置为200 - 250微米，以适应切片凸点和曲线。取决于Z-堆叠范围和组织的尺寸，每个全肺扫描将需要7之间 - 12小时用20X物镜。每个肺是唯一的，并且设置必须进行调整，每个实验。 </li></ol> PCLS 5.活细胞成像<ol><li> PCLS的30分钟温育的抗体后，从培养皿的抗体溶液中，用1毫升冷的Leibovitz-10冲洗切片两次。 </li><li>移液管50的新鲜的LeibovitzμL-10成一个一个腔玻璃罩（8个时隙）的时隙。用铲子将PCLS转印至介质，轻轻地操纵它，直到片是扁平的而展开。上述程序需要迅速执行，以避免光漂白。 </li><li>除去使用移液管的50μL的平台。切片要被躺在尽可能平坦。 注意:如果切片的边缘抵靠腔室的壁垂直向上翻转这是可接受的。此外，如果可用的话，金属铂的重量可用于在进行下一步骤之前，以锚定条带。见所附的材料清单为铂线的一个例子，可以被切割和弯曲成小的权重。 </li><li>在4℃冷室中，混合100微升生长因子减少，用100μL的Leibovitz-10不含酚红的细胞外基质的。使用预冷的吸头用于此，或细胞外基质将在尖端凝固。 注:基质和介质的这一比率（1:1）产生足够的致密保持PCLS下在组织培养条件的凝胶。 </li><li>轻轻吸取基质混合，并认真吸取在PCLS的顶部，确保凝胶那张露顶纳克片，并且所述片不会在凝胶的顶部上浮。凝胶应该工作作为权重，下锚定PCLS到板的底部。 </li><li>小心地将腔玻璃罩转移到37℃培养箱并让基质固化〜5分钟。 </li><li>传送整个腔室盖玻片到共焦显微镜的预热阶段。保持在整个成像37℃的室中。使用Leibovitz的介质时，不将细胞培养过程中所需的CO 2的供应。 </li><li>设定基于帧之间的所需间隔的z堆栈范围和瓦范围内。当共焦显微镜下观察，使用20X物镜（参见材料列表）。使用"瓦片"工具来表示的在组织中感兴趣的区域，使用"中心网格"设置（ 例如 ，2×2网格为中心的）。 </li><li>而设置的z堆栈范围，验证在切片所设置的范围是适当的多个区域。 注:ZR安格将可能等于组织的厚度。每个肺是唯一的，并且设置必须进行调整，每个实验。 2×2的瓷砖和10的z堆叠通常将导致〜1帧/ 2分钟。确保自动对焦功能开始在实验过程中成像期间以最小化的xy和z漂移之前被激活。 </li></ol>

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allergic+sensitization+through+the+airway+primes+Th17-dependent+neutrophilia+and+airway+hyperresponsiveness.">Allergic sensitization through the airway primes Th17-dependent neutrophilia and airway hyperresponsiveness.</a> Am J Respir Crit Care Med. 180 (8), 720-730 (2009).</li><li>Schlitzer, 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=Identification+of+cDC1-+and+cDC2-committed+DC+progenitors+reveals+early+lineage+priming+at+the+common+DC+progenitor+stage+in+the+bone+marrow.">Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.</a> Nat Immunol. 16 (7), 718-728 (2015).</li><li>Baluk, 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=Preferential+lymphatic+growth+in+bronchus-associated+lymphoid+tissue+in+sustained+lung+inflammation.">Preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation.</a> Am J Pathol. 184 (5), 1577-1592 (2014).</li><li>Jurisic, G., Iolyeva, M., Proulx, S. T., Halin, C., Detmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymus+cell+antigen+1+(Thy1,+CD90)+is+expressed+by+lymphatic+vessels+and+mediates+cell+adhesion+to+lymphatic+endothelium.">Thymus cell antigen 1 (Thy1, CD90) is expressed by lymphatic vessels and mediates cell adhesion to lymphatic endothelium.</a> Exp Cell Res. 316 (17), 2982-2992 (2010).</li><li>Truman, L. 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=ProxTom+lymphatic+vessel+reporter+mice+reveal+Prox1+expression+in+the+adrenal+medulla,+megakaryocytes,+and+platelets.">ProxTom lymphatic vessel reporter mice reveal Prox1 expression in the adrenal medulla, megakaryocytes, and platelets.</a> Am J Pathol. 180 (4), 1715-1725 (2012).</li><li>Wigle, J. T., Oliver, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+function+is+required+for+the+development+of+the+murine+lymphatic+system.">Prox1 function is required for the development of the murine lymphatic system.</a> Cell. 98 (6), 769-778 (1999).</li></ol>

作者有没有利益冲突申报。

这里描述的协议最初开发可视化肺内疾病预防控制中心两个子集的位置。然而，该协议可以很容易地适合于研究许多不同细胞类型，同时保持细胞活力和肺的三维结构。后者的特征是一个重要的优点在细胞培养系统，并促进稀有细胞类型的标识。该方法依赖于从肺PCLS的产生，和抗体的适当组合，以确定特定的细胞类型，同时最小化背景染色。在很大程度上，这是一个经验的运动，因为，在其他应...

以下促炎性刺激物的吸入，如脂多糖（LPS），有免疫细胞的协调运动成，内，并从肺。例如，嗜中性粒细胞迅速募集至肺实质及气道。此外，被称为常规树突细胞（cDC上）的一些专门的抗原呈递细胞进行相对复杂的迁移模式1，2。 cDC上可以使用流式细胞术来鉴定，部分地基于它们的表面标记的显示，CD11c的上。 DC的不同子集可以通过CD103和CD11b 3的差动表面表达来区分。当获取吸入抗原，一些cDC上退出肺并通过淋巴管迁移到肺引流淋巴结（LN中），其中它们呈现的肽抗原特异性T细胞4。这是在适应性免疫R的起始的关键早期事件esponses。不知什么原因，然而，并非收购吸入抗原所有cDC上离开肺，和许多这些细胞保持在该机构数月5，6。该观察可以通过将这些细胞的发育祖先可以部分地解释，因为单核细胞衍生的CD11c +缺乏趋化因子受体，CCR7的细胞，无法迁移到区域性淋巴结7,8。这很可能是疾病预防控制中心迁移潜力还确定，至少部分地由肺内的解剖位置。然而，在肺cDC上的这些不同的群体的精确定位不是完全表征。免疫细胞定位的肺中，并引导其分子的一种改进的知识，是需要更好地了解肺的免疫系统变得如何激活。 PCLS正在增加LY用来作为显现细胞的定位和细胞-细胞相互作用的离体方法中，同时维持肺结构9，10的结构完整性。 PCLS已用于研究许多物种，包括老鼠，牛，猴，羊，马和人11的肺。该技术的主要优点是，大约20片可以从小鼠肺的单个叶来制备，由此减少所需要的个体实验动物的数量。实际上，所有的免疫细胞类型（包括DC），巨噬细胞，中性粒细胞和T细胞，存在于PCLS和维持其正常结构。 PCLS也可以用来研究钙信号传导与气道和平滑肌细胞收缩与乙酰胆碱12或乙酰甲胆碱13处理之后。在这种方法中，只有肺的一小部分是一个微观alyzed，但一个研究报告指出，在PCLS气管收缩的测量从片仅改变约10％至切片，并且该差异是比得上在完整动物14使用肺功能测试看出。其他研究者使用PCLS作为离体方法用LPS 15温育后，研究在细胞因子的表达和细胞表面标志物的变化。 PCLS也已在小腺泡内动脉缺氧性肺血管收缩的离体模型中使用。这些容器位于不能使用其它的程序，包括从解剖动脉区段或胸膜下的血管16的分析记录到达肺的一部分。我们的实验室已主要用于PCLS可视化在稳定状态中活肺组织免疫细胞定位和以下的体内炎症刺激。这些都是我们的这个发展的过程如下。

<table><tbody><tr><td>C57BL/6J mice</td><td>Jackson Laboratory</td><td>000664</td><td></td></tr><tr><td>Prox1-TdTomato transgenic mice&amp;nbsp;</td><td>Jackson Laboratory</td><td>018128</td><td>B6;129S-Tg(Prox1-tdTomato)12Nrud/J</td></tr><tr><td>OT-II OVA-specific TCR x Nur77-GFP transgenic mice</td><td>Jackson Laboratory</td><td>004194, 016617</td><td>B6.Cg-Tg(TcraTcrb)425Cbn/J x C57BL/6-Tg(Nr4a1-EGFP/cre)820Khog/J&amp;nbsp;</td></tr><tr><td>Rag1 knock-out mice</td><td>Jackson Laboratory</td><td>002216</td><td>B6.129S7-Rag1&lt;sup&gt;tm1Mom&lt;/sup&gt;/J</td></tr><tr><td>Ovalbumin, Low Endo, Purified</td><td>Worthington Biochemical Corporation</td><td>LS003059</td><td></td></tr><tr><td>Lipopolysaccharides from Escherichia coli</td><td>Sigma-Aldrich Co.</td><td>L2630-25MG</td><td></td></tr><tr><td>Polyethylene tubing (Non-Sterile) 100 ft</td><td>BD Diagnostic Systems</td><td>427421</td><td>0.86 mm inside diameter, 1.27 mm outside diameter</td></tr><tr><td>GeneMate Sieve GQA Low Melt Agarose</td><td>BioExpress</td><td>E-3112-125</td><td>2% solution dissolved in PBS at 70 &amp;deg;C and held at 40 &amp;deg;C.</td></tr><tr><td>Compresstome VF-300</td><td>Precisionary Instruments, Inc.</td><td>VF-300</td><td></td></tr><tr><td>Double Edge Stainless Razor Blade</td><td>Electron Microscopy Sciences</td><td>72000</td><td>Disposable; 250/box. Blade should be changed for every lung.</td></tr><tr><td>Krazy Glue All Purpose Instant Gel</td><td>VWR</td><td>500033-484</td><td>Commonly available for $3/tube in local drugstores&amp;nbsp;</td></tr><tr><td>Leibovitz&#039;s L-15 Medium, no phenol red</td><td>ThermoFisher Scientific</td><td>21083027</td><td></td></tr><tr><td>Normal Rat Serum (NRS)</td><td>Jackson ImmunoResearch Inc.</td><td>012-000-120</td><td></td></tr><tr><td>Normal Mouse Serum (NMS)</td><td>Jackson ImmunoResearch Inc.</td><td>015-000-120</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Hyclone</td><td>SH30071.03HI</td><td></td></tr><tr><td>Staining Buffer</td><td>Made in House</td><td>N/A</td><td>PBS w/ 0.5% bovine serum albumin, 0.1% NaN&lt;sub&gt;3&lt;/sub&gt;, pH 7.4</td></tr><tr><td>Fc Blocker (anti-CD16/32 antibodies)</td><td>Made in House</td><td>N/A</td><td>Supernatant of cultured hybridoma cell line 2.4G2</td></tr><tr><td>Anti-mouse CD11b eFluor 450</td><td>eBioscience</td><td>48-0112-80</td><td>Anti-mouse CD11b eFluor 450 (clone: M1/70)&amp;nbsp;</td></tr><tr><td>Anti-mouse CD11c Brilliant Violet 605</td><td>BioLegend</td><td>101237</td><td>Brilliant Violet 605 anti-mouse CD11c (clone: M1/70)</td></tr><tr><td>Anti-mouse CD11c Phycoerythrin</td><td>eBioscience</td><td>12-0114-82</td><td>PE conjugated anti-mouse CD11c (clone: N418)</td></tr><tr><td>Anti-mouse CD11c Allophycocyanin</td><td>BD Phamingen</td><td>550261</td><td>APC-labeled anti-mouse CD11c 9clone: HL3)</td></tr><tr><td>Anti-mouse CD88 Phycoerythrin</td><td>BioLegend</td><td>135806</td><td>PE anti-mouse CD88 (clone: 20/70)</td></tr><tr><td>Anti-mouse CD103 Allophycocyanin</td><td>eBioscience</td><td>17-1031-82</td><td>Anti-mouse CD103 APC (clone: 2E7)</td></tr><tr><td>Anti-mouse CD90.2/Thy1.2 eF450</td><td>eBioscience</td><td>48-0902-82</td><td>Anti-mouse CD90.2 eFluor 450 (clone: 53-2.1)</td></tr><tr><td>Anti-mouse CD172a/Sirp1a Allophycocyanin</td><td>eBioscience</td><td>17-1721-82</td><td>Anti-mouse CD172a APC (clone: P84)</td></tr><tr><td>Anti-mouse CD324 Brilliant Violet 421</td><td>BD Horizon</td><td>564188</td><td>BV421 mouse anti-E Cadherin (clone: 5E8 also known as 5E8-G9-B4)</td></tr><tr><td>Anti-mouse CD324 Alexa Fluor 488</td><td>eBioscience</td><td>53-3249-82</td><td>Anti-CD324 (E-Cadherin) Alexa Flour 488 (clone: DECMA-1)</td></tr><tr><td>Anti-mouse CD324 Alexa Fluor 647</td><td>eBioscience</td><td>51-3249-82</td><td>Anti-CD324 (E-Cadherin) Alexa Flour 647 (clone: DECMA-1)</td></tr><tr><td>Glass Bottom Microwell Dishes 35 mm petri dish, 14 mm Microwell, No. 1.5 coverglass</td><td>MatTek Corperation</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass</td><td>ThermoFisher Scientific</td><td>155411PK&amp;nbsp;</td><td>Pack of 16</td></tr><tr><td>15 mm Coverslip, No. 1.5 Glass Thickness</td><td>MatTek Corperation</td><td>PCS-1.5-15</td><td></td></tr><tr><td>Bare Platinum Wire</td><td>World Precision Instruments</td><td>PTP201</td><td>0.020&quot; (0.5 mm) diameter cut into ~1 cm long pieces and bent into an &quot;L&quot; shape</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFisher Scientific</td><td>P36934</td><td>Keep at 4 &amp;deg;C, warm to room tempterature before use.</td></tr><tr><td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, *LDEV-Free</td><td>Corning</td><td>356231</td><td></td></tr><tr><td>Zeiss 880 multi-photon laser-scanning microscope</td><td>Carl Zeiss</td><td></td><td>Zen Black software version 8.1, 2012 (Zeiss)</td></tr><tr><td>Plan-Apochromat 20X/0.8 M27 objective lends</td><td>Carl Zeiss</td><td>420650-9901-000</td><td></td></tr></tbody></table>

precision-cut mouse lung slices to visualize live pulmonary dendritic cells

过敏原和病原体的吸入引起的各种肺免疫细胞类型的多个变化。流式细胞术是用于在免疫细胞上的细胞表面蛋白的定量分析的强大技术，但它提供对肺中这些细胞的定位和迁移模式的信息。类似地，趋化实验可以被执行以研究细胞的潜力， 在体外趋化因子反应，但是这些测定法不再现完整肺的复杂的环境。相对于这些前述技术中，肺中的个别细胞类型的位置可以容易地通过产生精确切割肺片（PCLS）中，用商购的，荧光标记的抗体染色它们，并通过共聚焦显微镜可视化部分可视化。 PCLS可以用于活的和固定的肺组织，和片可以涵盖区域一样大的整个叶的横截面。我们已经使用该协议成功地可视化的各种各样的肺细胞类型，包括不同类型的树突状细胞的巨噬细胞，嗜中性粒细胞，T细胞和B细胞，以及结构的细胞，例如淋巴，内皮的位置，和上皮细胞。可视化细胞相互作用，如树突状细胞和T细胞，在活的，三维的肺组织之间的能力，可以揭示细胞肺内如何移动，并与在稳定状态下和在炎症期间彼此相互作用。因此，与其他的方法，例如流式细胞术和定量PCR组合使用时，PCLS可以向背后肺的变应性和炎症性疾病的细胞事件的全面理解。

为了识别两个DC亚群从C57BL / 6小鼠中的位置，CD11b的喜 cDC上和CD103 + cDC上，PCLS切下并用特异于的CD11c单克隆抗体（mAb）染色，CD88，CD103，CD324和（E-钙粘蛋白）。抗体染色CD324气道上皮细胞，和CD88显示在巨噬细胞和嗜中性粒细胞，但不是cDC上8。这使我们能够从表面CD11c +巨噬细胞区分cDC上，并观察各细胞类型的气道（ 图1）</str...

Watch this Scientific Journal Video about Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells at JoVE.com

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

我们描述了用于产生精密切割肺片（PCLS）和免疫它们以显现各种免疫细胞类型的定位在肺中的方法。我们的协议可以扩展在各种条件下可视化的位置和许多不同细胞类型的功能。

精密切割的小鼠肺切片可视化现场肺树突状细胞

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for ...

precision-cut-mouse-lung-slices-to-visualize-live-pulmonary-dendritic

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

16.8K 次观看.  National Institute of Environmental Health Sciences, NIH. 我们描述了用于产生精密切割肺片（PCLS）和免疫它们以显现各种免疫细胞类型的定位在肺中的方法。我们的协议可以扩展在各种条件下可视化的位置和许多不同细胞类型的功能。 

视频: Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

科研

癌症研究

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

小鼠肺树突状细胞的分离

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

免疫与感染

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进<em&gt;在原位</em灌注肺组织和X-gal染色<em&gt; lacZ的</em&gt;标记的肿瘤细胞

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

医学

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

生物学

Human Lung Dendritic Cells: Spatial Distribution and Phenotypic Identification in Endobronchial Biopsies Using Immunohistochemistry and Flow Cytometry

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In order to maintain tolerance or to induce an immune response, the immune system must appropriately handle inhaled antigens. Lung dendritic cells (DCs) are essential in maintaining a delicate balance to initiate immunity when required without causing collateral damage to the lungs due to an exaggerated inflammatory response. While there is a detailed understanding of the phenotype and function of immune cells such as DCs in human blood, the knowledge of these cells in less accessible tissues, such as the lungs, is much more limited, since studies of human lung tissue samples, especially from healthy individuals, are scarce. This work presents a strategy to generate detailed spatial and phenotypic characterization of lung tissue resident DCs in healthy humans that undergo a bronchoscopy for the sampling of endobronchial biopsies. Several small biopsies can be collected from each individual and can be subsequently embedded for ultrafine sectioning or enzymatically digested for advanced flow cytometric analysis. The outlined protocols have been optimized to yield maximum information from small tissue samples that, under steady-state conditions, contain only a low frequency of DCs. While the present work focuses on DCs, the methods described can directly be expanded to include other (immune) cells of interest found in mucosal lung tissue. Furthermore, the protocols are also directly applicable to samples obtained from patients suffering from pulmonary diseases where bronchoscopy is part of establishing the diagnosis, such as chronic obstructive pulmonary disease (COPD), sarcoidosis, or lung cancer.

Lung-resident immune cells, including dendritic cells (DCs) in humans, are critical for defense against inhaled pathogens and allergens. However, due to the scarcity of human lung tissue, studies are limited. This work presents protocols to process human mucosal endobronchial biopsies for studying lung DCs using immunohistochemistry and flow cytometry.

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

The lungs are constantly exposed to the external environment, which in addition to harmless particles, also contains pathogens, allergens, and toxins. In ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Advanced Imaging of Lung Homing Human Lymphocytes in an Experimental In Vivo Model of Allergic Inflammation Based on Light-sheet Microscopy

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an attractive therapeutic target in the clinical management of affected patients. In order to further optimize strategies aiming at therapeutic regulation of pathologically imbalanced tissue infiltration of pro-inflammatory immune cells, it will be of particular importance to achieve improved insights into disease- and organ-specific homing properties of peripheral lymphocytes. The here described experimental protocol allows to monitor lung accumulation of fluorescently labeled and adoptively transferred human lymphocytes in the context of papain-induced pulmonary inflammation. In contrast to standard in vitro assays frequently used for the analysis of immune cell migration and chemotaxis, the now introduced in vivo setting takes into account lung-specific aspects of tissue organization and the influence of the complex inflammatory scenario taking place in the living murine organism. Moreover, three-dimensional cross-sectional light-sheet fluorescence microscopic imaging does not only provide quantitative data on infiltrating immune cells, but also depicts the pattern of immune cell localization within the inflamed lung. Overall, we are able to introduce an innovative technique of high value for immunological research in the field of chronic inflammatory lung diseases, which can be easily applied by following the provided step-by-step protocol.

The here introduced protocol allows characterization of the lung homing capacity of primary human lymphocytes under in vivo inflammatory conditions. Pulmonary infiltration of adoptively transferred human immune cells in a mouse model of allergic inflammation can be imaged and quantified by light-sheet fluorescence microscopy of chemically cleared lung tissue.

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

Overwhelming tissue accumulation of highly activated immune cells represents a hallmark of various chronic inflammatory diseases and emerged as an ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

精密切肺切片作为 离体 肺血管结构和收缩力研究的有效工具

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

使用琼脂糖包埋的振动切片机进行创新的肺部成像

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

作者聚焦:使用琼脂糖包埋的肺组织切片研究空间蛋白表达 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

精密切割的小鼠肺切片可视化现场肺树突状细胞

摘要

探索更多视频

此视频中的章节

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

小鼠肺树突状细胞的分离

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

精密切肺切片作为离体肺血管结构和收缩力研究的有效工具

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

精密切割的小鼠肺切片可视化现场肺树突状细胞

摘要

探索更多视频

此视频中的章节

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

小鼠肺树突状细胞的分离

肺转移瘤在小鼠体内的组合在单细胞分辨率的可视化改进

标准化的制备小鼠肺组织单细胞悬浮液使用gentleMACS Dissociator

人肺树突状细胞:空间分布与表型鉴定支气管活检采用免疫组化和流式细胞仪

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

基于光片显微镜的变应性炎症体内模型中肺呼名人淋巴细胞的高级成像

精密切肺切片作为 离体 肺血管结构和收缩力研究的有效工具

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

精密切肺切片作为离体肺血管结构和收缩力研究的有效工具