我们感谢来自博士的讨论，指导和支持。迈克尔Andreeff博士。，和Jared伯克斯博士。从MD安德森流式细胞仪和细胞成像核心设施。这项工作是由美国国家癌症研究所（RC1-CA146381，CA-083639，R01NS06994，CA116199和CA109451对于FCM部分资金。ELS是由国防部陆军部（BC083397）也支持。

同基因小鼠骨髓移植（BMT）批准的机构IACUC协议（ 注:此协议的成功需要利用任何标记的细胞类型（S）的为多光谱的分析目标，并促进下游分析，人们可以利用任何遗传或稳定的细胞标记，我们建议的任何细胞，组织或器官将要能够应用于移植模型，而移植可以包含尽可能多的独特标记的种群为研究认为是有用的。此外，多重标记的蜂窝系统，如在转基因小鼠中发现包含多个谱系限制，荧光标记的人群，可以设计，以消除移植并发症的某些病理状态的研究。 <ol><li>致死量照射骨髓移植受体小鼠的9.5戈瑞四个小时单剂量重建与捐助骨髓前。 （GFP骨髓移植受者应该是6-10周龄）。 </li><li>剪切，剥离回暴露后肢用无菌手术剪之前弄湿鼠标皮用70％乙醇。删除整个腿部，包括髂嵴在骶髂关节。通过削减略高于脚踝，然后从骨头彻底清除所有的肌肉，韧带和多余的组织丢弃的脚。冲洗骨髓从胫骨，fibia和髂嵴与使用2％无菌胎牛血清的PBS（PBS-2）一23号针头。 （BMT RFP捐助者应根据机构的标准被牺牲。） </li><li>片，并轻轻挤压剩余骨成使用手术刀和镊子的片段，然后放入50ml锥形管中与3微克/毫升胶原酶我1小时，在37℃，转速300rpm。 </li><li>顶过管子用PBS-2和收集上清到一个新的试管，并结合冲洗细胞。降速以250rpm的转速为5分钟，在4°C。 </li><li>重悬细胞于PBS-2，并通过40纳米细胞过滤器过滤。在这一点上，该细胞可以被标记为fluorescen吨激活细胞分选（FACS）如果一个人想操纵的BMT特定人群。 </li><li>重悬，每100微升PBS 2×10 6个细胞，每只小鼠。使用29号针头由尾部或眶后静脉斜角朝上系统地注入绿色荧光蛋白照射受体小鼠。注入一个鼠标用100微升PBS仅作为植入控制。用热灯前注射扩张血管和注射后用尾静脉注射克制。有无菌纱布可应用于光压到尾后注入。 </li><li>三周后BMT，收集血液眼窝并通过流式细胞术分析，以确认GFP的循环细胞的存在和不存在的RFP循环细胞。控制鼠标会在这一点上已经死亡。 </li></ol> 2。肿瘤移植植入据机构IACUC准则<ol><li>成长ID8 体内注射液（1×10 6 C前卵巢鼠的肿瘤细胞在体外每只小鼠的ELL）。注射的当天，胰蛋白酶消化细胞，用PBS洗涤，用PBS重悬在每100μl的1×10 6个细胞。 </li><li>注射肿瘤细胞到小鼠的intaperitoneal腔，针应当斜面朝上。灭菌注射的视线用70％乙醇。注入左下或腹部右下象限（颅稍内侧到下集的雌性小鼠腹部乳头），避免撞上器官。由颈背抓握住尾巴与小指或无名指抑制小鼠。小鼠可以用异氟烷麻醉用于此过程。 </li><li>牺牲小鼠时肿瘤细胞植入的迹象，比如轻微腹胀，是显而易见的。这将发生在4-12个星期。 </li><li>消费肿瘤来自IP腔。在腔内肿瘤的样子就和周围器官的表面的白色结节。用解剖刀，取出肿瘤并放置在10％福尔马林24小时固定的管/罐。组织学p赔偿可以外判予病理学/组织学的核心，如果试剂和材料不容易获得的石蜡包埋和幻灯片的准备。部组织成5-8微米切片载玻片上用于随后的染色过程。 </li></ol> 3。多参数免疫荧光染色<ol><li>准备单色控制幻灯片的每个荧光探针在实验中使用。在这种情况下，四种颜色的使用。因此，四张幻灯片为每个单独的颜色以及背景噪声的控制和完整的组合染色最后一张幻灯片一张幻灯片。 （共载玻片= 6）所有的幻灯片将被处理并排以相同的方式，以减少实验误差。 </li><li>烤石蜡切片，在56℃下反应1小时。 </li><li>洗涤载玻片3倍10分钟，二甲苯等。 </li><li>洗2×5分钟，100％乙醇中。 </li><li>洗涤1X 3分钟95％的乙醇</li><li>洗涤1X 2 min90％的乙醇。 </li><li>洗涤1X 2 min70％的乙醇。 </li><l我&gt;洗1X 2分钟水。慢慢地浸泡染色架在水中的10倍进出。 </li><li>在最后一次洗涤系列，2分钟（微波高）预煮柠檬酸钠缓冲液。 （可以用Tris EDTA缓冲液根据所使用的抗体取代） </li><li>在柠檬酸钠缓冲液（微波对10％的电力，或在高压锅）煮沸的幻灯片20分钟。如果缓冲区沸腾，关掉微波炉，让坐。 </li><li>从热源取出，放幻灯片休息30分钟，在柠檬酸钠缓冲液降温。 </li><li>冲洗载玻片在水中5分钟。 </li><li>周围的肿瘤切片用巴氏钢笔标记，并把2％BSA / 1％FBS的马来酸的封闭缓冲液1小时。单独准备的幻灯片，以不让他们在这一步干出来的。 </li><li>在封闭缓冲液中制备的初级抗体在最佳稀释。 （如果使用的抗体是所有不同种类的（或IgG链的变化，例如:IgG1的对同一物种的IgG2a的），他们可能会期间作为组合使用英格尔潜伏期。如果抗体物种重叠，然后孵育，必须依次进行，以减少抗体的交叉反应。） </li><li>在这一点上，留下两个幻灯片，只有封闭液。这两个幻灯片将作为未染色的对照和核染色控制。其他单一色彩控制将孵育个别主要抗体。只有在分析滑动将具有第一抗体的结合。 </li><li>孵育在在上缓慢旋转摇床的水分腔框幻灯片与第一抗体4℃过夜（〜16-20小时）（ 注:带4 +的荧光团，连续染色法或直接缀合工作时可能是必要的，以减少抗体的交叉反应在这情况下重复步骤1.16-1.21直到所有的抗体应用于幻灯片。） </li><li>在PBST 10分钟（2个）轻轻振动筛洗幻灯片</li><li>虽然洗是怎么回事，准备Alexafluor 488，594和647的二抗的最终稀释在封闭缓冲液中1:1000。 （确保这些抗体和稀释液保存在4℃，并在黑暗中在任何时候都保持荧光跨批次和时间的推移）。 </li><li>在单色控制幻灯片，放置单个Alexafluor稀释匹配使用的主要抗体的物种。所有的二抗将被放置在分析滑动。未染色和核深染控制幻灯片将继续留在封闭液经过该步骤。 </li><li>孵育在缓慢旋转振荡器在室温下2小时的保湿室箱（用铝箔覆盖，避免光线照射）。 </li><li> 5分钟在PBST（2个）轻轻振动筛洗幻灯片。盖上铝箔避光。 </li><li>加DAPI（1:10,000），持续1分钟到DAPI只幻灯片和分析滑动 。盖上铝箔避光。其他幻灯片可能会留​​下覆盖在洗涤容器。 </li><li>洗两DAPI孵育在第5分钟，洗涤，以不会污染其他幻灯片用DAPI染色单独的容器中滑动。对于第二个5分钟的洗涤，幻灯片可以在PBST中进行组合（所有洗涤液应在振荡器上进行的）。盖上铝箔避光。 </li><li>在水中浸简要滑梯前盖片（1.5厚度与水的安装介质保存荧光） </li><li>让干燥3小时，在黑暗中在室温。 </li><li>密封盖玻片用指甲硬化剂的边缘。 （不要使用指甲油的丙酮可淬的荧光信号）。 </li><li>立即分析幻灯片或在4℃保存，供日后分析。 </li></ol> 4。多光谱成像<ol><li>收集的OLYMPUS X-63正置显微镜与Nuance的软件，用于数据收集利用Nuance的EX摄像机（含液晶可调谐滤波器）多光谱影像。 </li><li>使用油浸物镜来提高分辨率搜集图像。 </li><li>创议l设置了包括设置光谱范围为每个使用中的荧光团。 <ol class="lalpha"><li>对于4色: <ol class="lroman"><li> DAPI:420-480nm波长</li><li> AF488:490-580nm范围内</li><li> AF594:590-640nm的</li><li> AF647:650-720nm以下</li></ol></li></ol></li><li>取分析滑动的初始图像，以获得适当的曝光时间对于每个光谱范围内的。 </li><li>创建"光谱库"，该软件将用来识别和区分彼此的信号与噪声的荧光团的比例和背景信号。 （ 注:每个荧光染料必须有自己的单一色彩控制幻灯片以组装与适当波长曲线的光谱库，以确保数据集的正确分析举例:3荧光= 4控制幻灯片; 5荧光= 6控制幻灯片）。 <ol><li>图像的未染色的幻灯片第一。这张幻灯片将频谱图书馆，A内指定Ÿ为背景 。&gt; </li><li>图像的DAPI只滑动秒。这张幻灯片将用于指定DAPI染色细胞核与"画"的工具，将谱库为DAPI内保存。&gt; </li><li>图像的AF488只滑动三分之一。这张幻灯片将用于指定AF488染色区域与"画"的工具，将谱库为AF488内保存。&gt; </li><li>图像的AF594只滑第四名。这张幻灯片将用于指定AF594染色区域与"画"的工具，将谱库为AF594内保存。&gt; </li><li>图像的AF647只滑第五名。这张幻灯片将用于指定AF647染色区域与"画"的工具，将谱库为AF647内保存。&gt; </li><li>下列各个频谱分量的集合，保存该谱库和协议。&gt; </li></ol></li><li>一旦谱库完成后，找到自己感兴趣的区域上的显微镜分析幻灯片 ，并收集/保存图像。&gt; </li></ol> 5。多光谱图像的定量分析<ol><li>导入使用的InForm软件收购Nuance的图像的代表品种。 </li><li>打开保存的谱库中所指示的程序</li><li>确定感兴趣的组织区域进行分析。 （ 如肿瘤组织与脂肪组织）。 </li><li>确定基于DAPI染色的单个细胞。细胞质将自动基于所述核的形状来限定。 </li><li>挑荧光强度读出所选择的每个荧光参数（ 如平均值，标准偏差） </li><li>一批采集的图像，并让使用该算法的软件过程。 </li><li>当图像处理结束后，合并结果文件。 </li><li>打开结果在Excel（或任何其他分析软件）和暗算对方两种荧光强度的XY轴。各荧光强度每单元给出。的双阳性细胞将出现在右上象限。用户必须定义​​基线荧光强度。 </li></ol>

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FCM，招商银行和ELS有没有冲突，并没有透露。

本文中我们描述了多光谱成像的应用中，我们描述了作为复用的细胞组合物MIMicc-多光谱询问，以分析肿瘤微环境中的源自骨髓的间质成分，但是这种方法和概念可以应用于破译了组成一个复杂的其他细胞成分组织中，例如在伤口愈合反应或再生组织中看到。在这些实验中，我们利用转基因小鼠中，以荧光从内植入肿瘤微环境中的宿主来源的细胞区分骨髓来源的细胞，但该技术适合于使用任何报告基因标记的细胞，并且特别易于进行细胞表达基因的标签很容易被免疫组化可辨，如LacZ基因，葡萄糖醛酸酶，他（6X） - HA，FLAG标签标记的细胞， 等 11。 在我们的模型中，继移植小鼠的牺牲，肿瘤切片处理，染色的抗体，以确定细胞的来源;源性RFP +骨髓捐赠者或GFP +宿主组织来源。在图2中，我们展示两个标记共表达用宿主衍生的GFP +肿瘤微环境内的细胞。然而，我们显示了使用相同的系统在图2D 7色染色的潜力。 这些数据是对什么是能够利用我们的移植及多光谱成像模型的表示。这个系统是非常灵活的，可以容纳更多的参数，包括:1，改变了骨髓移植的人群/转基因小鼠模型，2 -交替肿瘤/疾病模型，或3 -备用标记（ 如表面，细胞内，磷酸化蛋白质）。通常情况下，用同一张幻灯片上提高免疫组织化学染色的数字，数据分析变得艰巨，但是用多光谱成像平台和阿索ciated混溶软件可实现高效，可靠和可重复的结果。我们提供了潜在的抗体种类，并在表1荧光标记物的组合。此外，该技术允许因为使用这两种冷冻和石蜡包埋的组织切片使得这种技术适用于除了基本的研究模型临床组织样本的潜在的多功能性在探针/抗体选择。

了解组织的发育和修复是显著，以阐明在伤口愈合，2,3再生医学，发育生物学，肿瘤生物学参与细胞成分。正在修理的情况下，许多类型的细胞渗透到周围的微环境中血管生成，细胞外基质沉积，增殖和组织结构调整提供帮助。细胞因子和表型可以基于多参数，复用标记，可以识别定位，分化状态和所研究的微环境中的细胞成分之间的相互作用来识别。在这里，我们描述肿瘤的发展作为一个典型的例子为这个多色多细胞移植模型其次是多光谱成像和光谱分离的方法。 肿瘤进展是显着的几个收购功能，包括增强的扩散，一个多步骤的过程ntiapoptotic，浸润和血管生成特性。4肿瘤的发展是由被招募到周围环境中，以提供生长因子，结构基质，血管网络和免疫调节非肿瘤细胞容易。1,5,6这种微生包括衍生自细胞的本地，邻近的组织，例如脂肪，血管和遥远来源如骨髓衍生细胞1。非赘生性细胞结合的程度依赖于来自肿瘤，这往往对应于肿瘤的阶段/级的需求。理解肿瘤微环境中支持的作用，人们必须了解的非肿瘤细胞群的起源和分化潜能。 这个协议被设计在肿瘤进展的同时通过骨髓来源的细胞组分可视化的解释，以帮助和局部组织衍生工编细胞。利用荧光报告基因表达的转基因小鼠，我们移植GFP（绿色荧光蛋白）的骨髓成致死剂量照射的RFP（红色荧光蛋白）鼠标。继成功的骨髓移植，同基因肿瘤细胞系原位注入并允许嫁接4-8个星期。将所得的肿瘤是从小鼠切下并处理用于免疫荧光（IF）染色进行可视化的基质组分。复中频标记是一种常用的技术，它涉及显著优化7-9，不过使用的是多光谱成像/解混平台提高对于具有光谱重叠的荧光标记物的组合的可能性。在此，我们提出我们称之为复用蜂窝组成MIMicc，多光谱审讯染色及分析多达内对单张幻灯片肿瘤部分8标志物，以分析细胞起源，细胞分化ST的技术ATUS和元器件的肿瘤微环境内的细胞 - 细胞相互作用。这个简化的例子有在为了分析五个，六个或更多个标记物，利用抗体或细胞内的启动子驱动的荧光表达， 表1列出潜在荧光抗体染色组合以考虑相应的物种被扩展的可能性。

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td></td>
		 </tr>
		 <tr>
			<td>.2 &mu;m filter</td>
			<td >Fisher</td>
			<td>09-740-35A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml lure lock </td>
			<td >BD</td>
			<td>309604</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 G needle</td>
			<td >Cardinal</td>
			<td>BF305122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>40nm filter</td>
			<td >BD</td>
			<td>352340</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml conical tube</td>
			<td >Fisher</td>
			<td>1495949A</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 488-ms1</td>
			<td >invitrogen</td>
			<td>A21121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 594-Rb</td>
			<td >invitrogen</td>
			<td>A21207</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Alexafluor 647-ch</td>
			<td >Invitrogen</td>
			<td>A21449</td>
			<td></td>
		 </tr>
		 <tr>
			<td>BSA</td>
			<td >Sigma</td>
			<td>A7906-500G</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Cover slips</td>
			<td >Corning</td>
			<td>2940-243</td>
			<td></td>
		 </tr>
		 <tr>
			<td>CRI-Nuance</td>
			<td>Caliper Life Sciences</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dapi</td>
			<td >Invitrogen</td>
			<td>D1306</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DMEM</td>
			<td >CellGro</td>
			<td>10-017-CV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol</td>
			<td >Dharmacon</td>
			<td>4004-DV</td>
			<td></td>
		 </tr>
		 <tr>
			<td>FBS</td>
			<td >invitrogen</td>
			<td>16000-044</td>
			<td></td>
		 </tr>
		 <tr>
			<td>GFP-ms1</td>
			<td >Abcam</td>
			<td>ab38689</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Insulin needle</td>
			<td >BD</td>
			<td>329424</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Maleic acid</td>
			<td >Acros</td>
			<td>100-16-7</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Moisture chamber box</td>
			<td >Evergreen</td>
			<td>240-9020-Z10</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mounting media</td>
			<td >dako</td>
			<td>S3023</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nail hardener</td>
			<td >Sally Hansen</td>
			<td>2103</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pap pen</td>
			<td >Abcam</td>
			<td>Ab2601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pen/Strep L Glut</td>
			<td >invitrogen</td>
			<td>10378016</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Steril PBS</td>
			<td >invitrogen</td>
			<td>14040-182</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin</td>
			<td >invitrogen</td>
			<td>252000-56</td>
			<td> </td>
		 </tr>
 <tr>
 	<td></td>
 <td></td>
 <td></td>
 <td>
 Blocking Buffer	
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td>maleic acid</td>
 <td>14.51 g </td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>10.95 g</td>
 </tr>
 <tr>
 <td>NaOH pellets (to 7.5pH)</td>
 <td>9+g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>250 ml</td>
 </tr>
 </tbody>
 </table>
	*add 0.2% Tween-20 to 1x solution for blocking buffer with 2%BSA/2%FBS. Stir and filter. 
	Antigen retrieval buffers:
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td colspan="2">Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0)</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>1.21 g</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>0.37 g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>1000 ml (100 ml to make 10x, 50 ml to make 20x)</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
 </table>
	*Store at RT for 3 months or at 4 &deg; C for longer
	<table border="1" fo:keep-together.within-page="always">
 <tbody>
 <tr>
 <td colspan="2">Sodium Citrate Buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0)</td>
 </tr>
 <tr>
 <td>Tri-sodium citrate (dihydrate)</td>
 <td>2.94 g</td>
 </tr>
 <tr>
 <td>Distilled water</td>
 <td>1000 ml</td>
 </tr>
 <tr>
 <td>HCl (adjust pH to 6.0)</td>
 <td>1N</td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>0.5 ml</td>
 </tr>
 </tbody>
 </table>
 </td>
 </tr>
		</tbody>
 </table>

quantitative multispectral analysis following fluorescent tissue transplant for visualization of cell origins, types, and interactions

以理解的多个蜂窝单元，以一种复杂的组织的发展贡献的愿望;如参与再生组织，肿瘤形成，或血管生成中的许多细胞类型中，我们设计了在肿瘤发展的一个多彩色的细胞移植模型该细胞群的不同颜色的荧光报告基因小鼠起源和移植，嫁接或和周围一个发展中的肿瘤注射。然后，这些彩色的细胞被招募并纳入肿瘤基质。为了定量评估骨髓来源的肿瘤基质细胞，我们移植绿色荧光蛋白转基因表达全骨髓成致死剂量照射RFP批准的IA​​CUC表达的小鼠。该被原位注入移植小鼠0ovarian切除肿瘤6-8周后移植和原籍骨髓标记（GFP）以及抗体标记物来检测肿瘤相关分析基质使用多光谱成像技术。然后，我们修改我们称之为复用蜂窝组成MIMicc - 多光谱讯问的方法，使用fluoroprobes的多光谱混合像元分解定量评估这些标记的细胞来自这起群体（以原价报告基因标签），并作为我们UNMIX 4,5能力，每张幻灯片增加6个或更多频谱，我们已经添加了细胞谱系或分化增加精度相关的其他免疫组化。利用软件来检测共定位复用的荧光信号，肿瘤间质的人群可追溯，枚举和特点的基础上标记染色。1

利用多光谱成像技术来分析肿瘤植入在我们的转基因骨髓移植小鼠模型，我们能够辨别间质瘤组件是骨髓来源。初始BMT证实3周移植后通过流式细胞术（ 图1）。原位注射未标记卵巢肿瘤BMT后确认植入切除和福尔马林固定的6周后最初的肿瘤注射。石蜡包埋的肿瘤切片，切片在&lt;8μm的切片和染色。适当的单一色彩控制幻灯片的每个荧光基团包括一个用于背景自发荧光而进行。 表1列出了潜在的实物和荧光组合2-7参数。继染色过程中，使用的是Nuance的相机附件，Nuance的软件进行采集的图像。创建谱库用单色控制幻灯片（ 图2A）后，我们瓦特的ERE能够"UNMIX"的拍摄的显示（抗体＃1）随着核的DAPI和其他两个标记GFP表达多重标记的卵巢肿瘤切片图像（抗体＃2和＃3）。 （ 图2B）加法的多种标记物是可能的，并且图2C-D示出了具有六个标志加上一个核染色肿瘤部分的图像。最后，所有采集的图像被导入的InForm（CaliperLS，霍普金顿，MA）软件进行分析。 Nuance公司（CaliperLS，霍普金顿，MA）软件与其他包一样的MetaMorph（Molecular Devices公司）或IPLab（Scanalytics，BD公司）兼容。此外，图像和光谱数据集，也可以保存为TIFF文件和读取该文件格式的任何程序中查看。使用的InForm软件，我们能够对感兴趣的肿瘤切片中的组织区域（ 图3A和E）进行分类，并设置算法的每个细胞浅各荧光染料的荧光强度版上的DAPI核染色（ 图3B-C）。最后，我们可以绘制基于两种标记物，AF488和AF647（ 图3D）的荧光强度的每个单独的单元格。我们建立了一个双阳性细胞群的基础上所确定的荧光强度的阈值（ 图4）。作为数据集变得更复杂具有多个参数，有潜在的用铲软件10（的密度归一化的事件生成树级数分析）来分析数据，尽管这尚未被执行。此软件可共表达趋势在大数据集的可视化，并且可以暗示集群分析组织切片内共表达标记之间的关联/关系。 表1中。 2-7彩色多重染色免疫荧光染色控制的准备。 （a）包括最多四色选项（B） </stroNG&gt;包括多达利用的违反光谱的近红外部分，而（C）包括可见光光谱内的七种颜色选项的抗体七色选项。 <img src="/files/ftp_upload/50385/50385fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50385/50385fig1highres.jpg"/> 图1。骨髓移植的确认通过流式细胞仪。 （A）中的GFP转基因受体小鼠为正（B）为RFP +造血细胞相比，控制RFP +和GFP +细胞控制的GFP +小鼠和阴性（C）内循环的造血细胞群的点积。 <img src="/files/ftp_upload/50385/50385fig2.jpg" alt="图2"/> 图2。未混合光谱图像处理。 （A） </stron，g&gt;了解卵巢肿瘤切片分析产生的4色光谱库的截图（二）多光谱图像显示荧光标记的细胞小鼠的卵巢肿瘤的部分;＃1:AF488（前495纳米/ EM 510纳米）;＃2 :AF594（ex590/em617纳米）;＃​​3:AF647（前650/em 668 nm）和核:DAPI（前350/em 470纳米）。单一颜色显示为白色的卵巢肿瘤切片分析创造了7彩光谱库中的（C）截图（D）多光谱图像显示荧光标记的细胞小鼠的卵巢肿瘤的部分;核:。DAPI（ex350/em470纳米）; AF488（前495/em 519纳米），AF514（ex518/em540纳米）; AF568（前578/em 603纳米），AF594（前590/em 617纳米），AF633（前632/em 647纳米）; AF660（前663/em 690 nm）和。单一颜色显示为白色，以及相匹配的复合色。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50385/50385fig2large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50385/50385fig3.jpg" alt="图3" fo:content-width="4.75in" fo:src="/files/ftp_upload/50385/50385fig3highres.jpg"/> 图3。定量MIMicc描写通知分析。 （一）首先形象代表组织分部其次是（B）基于核DAPI染色细胞的分割分类（C）荧光标记的定量可以在细胞核和细胞质中进行测量，并出口到（D） Excel作进一步分析。（五）组织的分割算法，能够进一步识别基于组织结构，而不是仅仅对荧光鉴定提供了更强大的分析工具，在不同的组织区域分类染色组织亚型。在此图像中，电脑已经被训练识别5分区域的基础上architectura组织内升图案:背景是由空格定义，溶血性肿瘤是由丰富的组织内红细胞定义，肿瘤是由人口密集的单元格定义，脂肪是由大的，对称的，空的细胞区室的定义，肌肉由条纹图案定义。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50385/50385fig3large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/50385/50385fig4.jpg" alt="图4"/> 图4。从20获得的图像的单个细胞的情节显示了基于GFP的荧光强度标有AF488和αSMA标有AF647的双阳性细胞。 补充文件。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50385/Tips_and_Tricks_for_Multiplexing_5_or_more_probes.docx">提示和技巧复用5个或更多的探针。</a> </a&gt;

Watch this Scientific Journal Video about Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions at JoVE.com

Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

复合组织群众，从机关到肿瘤，是由各种细胞成分的。我们阐明了利用多标记的荧光转基因小鼠与多参数免疫荧光染色结合组织随后光谱分离破译细胞起源以及基于蛋白表达细胞特征的细胞内表型的贡献。

多光谱定量分析继荧光组织移植的细胞起源，类型和相互作用的可视化

With the desire to understand the contributions of multiple cellular elements to the development of a complex tissue; such as the numerous cell types that ...

quantitative-multispectral-analysis-following-fluorescent-tissue

Nanyang Technological University

Research

JoVE Journal

Medicine

9.3K 次观看.  MD Anderson Cancer Center. 复合组织群众，从机关到肿瘤，是由各种细胞成分的。我们阐明了利用多标记的荧光转基因小鼠与多参数免疫荧光染色结合组织随后光谱分离破译细胞起源以及基于蛋白表达细胞特征的细胞内表型的贡献。 

视频: Quantitative Multispectral Analysis Following Fluorescent Tissue Transplant for Visualization of Cell Origins, Types, and Interactions

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

主动脉原位移植大鼠模型，研究慢性血管病变的发展

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

科研

医学

Quantitative Visualization and Detection of Skin Cancer Using Dynamic Thermal Imaging

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment for melanoma remains surgical excision, therefore, the key to extended survival is early detection 2,3. Considering the large numbers of patients diagnosed every year and the limitations in accessing specialized care quickly, the development of objective in vivo diagnostic instruments to aid the diagnosis is essential. New techniques to detect skin cancer, especially non-invasive diagnostic tools, are being explored in numerous laboratories. Along with the surgical methods, techniques such as digital photography, dermoscopy, multispectral imaging systems (MelaFind), laser-based systems (confocal scanning laser microscopy, laser doppler perfusion imaging, optical coherence tomography), ultrasound, magnetic resonance imaging, are being tested. Each technique offers unique advantages and disadvantages, many of which pose a compromise between effectiveness and accuracy versus ease of use and cost considerations. Details about these techniques and comparisons are available in the literature 4.
Infrared (IR) imaging was shown to be a useful method to diagnose the signs of certain diseases by measuring the local skin temperature. There is a large body of evidence showing that disease or deviation from normal functioning are accompanied by changes of the temperature of the body, which again affect the temperature of the skin 5,6. Accurate data about the temperature of the human body and skin can provide a wealth of information on the processes responsible for heat generation and thermoregulation, in particular the deviation from normal conditions, often caused by disease. However, IR imaging has not been widely recognized in medicine due to the premature use of the technology 7,8 several decades ago, when temperature measurement accuracy and the spatial resolution were inadequate and sophisticated image processing tools were unavailable. This situation changed dramatically in the late 1990s-2000s. Advances in IR instrumentation, implementation of digital image processing algorithms and dynamic IR imaging, which enables scientists to analyze not only the spatial, but also the temporal thermal behavior of the skin 9, allowed breakthroughs in the field.
In our research, we explore the feasibility of IR imaging, combined with theoretical and experimental studies, as a cost effective, non-invasive, in vivo optical measurement technique for tumor detection, with emphasis on the screening and early detection of melanoma 10-13. In this study, we show data obtained in a patient study in which patients that possess a pigmented lesion with a clinical indication for biopsy are selected for imaging. We compared the difference in thermal responses between healthy and malignant tissue and compared our data with biopsy results. We concluded that the increased metabolic activity of the melanoma lesion can be detected by dynamic infrared imaging.

We demonstrated that malignant pigmented lesions with increased metabolic activity generate quantifiable amounts of heat and the measurement of the transient thermal response of the skin to a cooling excitation allows quantitative identification of melanoma and other skin cancers (vs. non-proliferative nevi) at an early stage of the disease.

定量可视化和皮肤癌症使用动态热成像检测

In 2010 approximately 68,720 melanomas will be diagnosed in the US alone, with around 8,650 resulting in death 1. To date, the only effective treatment ...

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 ...

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

验尸报告组织心肌细胞核分离

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

在疾病的染色质蛋白质组的定量分析

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

小鼠异位心脏移植技术

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Murine Kidney Transplant Technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

小鼠肾脏移植技术

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

静脉介入型号:研究旁路移植通畅一个合适的模型

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

通过轻型显微镜对小鼠急性心肌炎模型中白细胞浸润的定量可视化

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...