作者要感谢Michael Easterling博士（社会和科学系统公司，达勒姆，NC），他对这种方法的验证和Tiago Ferreira博士（加拿大魁北克蒙特利尔的麦吉尔大学）持续不断的协助协助Sholl申请。

本研究的所有动物使用和手术均由NIEHS实验室动物护理和使用委员会批准，并在实验动物护理认证机构评估和认证协会进行。 消费乳腺<ol><li>使用二甲苯防止方法（铅笔效果最佳）预先标记所有的幻灯片。在末端覆盖安装解决方案以保护标签。 </li><li>通过机构动物保护和使用委员会批准的方法对动物进行安乐死。 </li><li>安乐死后，将动物放在解剖板上（ 图1 ）。拉伸并扣住所有四肢，后肢形成倒V形（保持针或小规格针工作良好）。 </li><li>用70％乙醇充分喷雾腹部，以保持头发脱离乳房样品。 </li><li> 使用镊子，拉起腹部皮肤在中线，并做一个小切口解剖剪刀，小心不要穿刺腹膜。 <ol><li>从切口开始，将皮肤切割至颈部区域，然后远离前肢。切割到腹股沟区域，然后向远侧方向延伸到同一侧后肢的第一关节;该切口通常分离乳腺5和6.​​避免切割腹膜。 </li><li>使用镊子将附着的乳房脂肪垫拉回皮肤，使用解剖剪刀的平头端将其从腹膜轻轻分离。单独作为远背侧尽可能以完全暴露4和第 5腹股沟乳腺在皮肤上的下侧;将皮肤固定在解剖板上（ 图2 ）。 </li><li>轻轻抓住第 5腺体的乳腺组织用弯钳的圆形侧慢慢地修剪腺远离皮肤，小心不要划伤腺或皮肤。 </li><li>继续修剪，直到第4和第 5 个腺体完全从皮肤上解离出来。确保乳头区域与腺体的其余部分一起被去除，因为它作为Sholl分析的起点。切割乳腺组织远离其附着于腺体的身体4。 </li></ol></li><li>一旦将腺体从动物中取出，将其均匀分布在带静电的25 mm x 75 mm x 1 mm显微镜载玻片上，边缘与皮肤相邻。 注意:对于老年或哺乳动物的腺体，可能需要51 x 75 x 1毫米的幻灯片。 </li><li>戴手套时，请轻轻挤出任何气泡。用塑料石蜡膜和另一个显微镜载玻片覆盖腺体，并压缩腺体以将其粘附到载玻片上。 注意:充满水的50mL锥形管适合用作重量。粘附到载玻片上所需的时间取决于腺体的厚度。薄，postna天天（PND）4腺应压缩至少30分钟，而较厚的成体腺体可能需要长达2-5小时。 </li></ol> 2.准备乳房整体<ol><li> 至少提前24小时准备胭脂红矾溶液，因为它需要煮沸和制冷。 <ol><li>将1克胭脂红明矾和2.5克硫酸铝钾（AlK（SO 4 ） 2 ·12H 2 O）溶解在500毫升蒸馏水中，并在1升烧瓶中煮沸20分钟。使用蒸馏水使最终体积达到500 mL。 </li><li>在真空下通过滤纸过滤溶液并冷藏保存。 注意:胭脂红矾溶液可以重复使用，但当颜色开始褪色时应该丢弃。 </li></ol></li><li>在固定前，将石蜡剥离腺体，小心不要将腺体从载玻片上拉出。将幻灯片放置在玻璃载玻片上，并将其浸入固定剂（100％乙烷）中醇，氯仿和冰醋酸，比例为6:3:1），取决于腺体的厚度。 </li><li>倾倒固定剂，并将腺体浸泡在70％乙醇中15分钟。通过倒出1/3的乙醇溶液并用蒸馏水代替，逐渐使腺体再次水化。浸泡5分钟重复此过程三次。 </li><li>最后冲洗后，倒出所有乙醇/水溶液，并用100％蒸馏水代替。浸泡腺体5分钟。 </li><li>倒出蒸馏水，将腺体浸入胭脂红矾溶液中。根据厚度，将腺体染色12-24小时。 注意:腺体不能过度染色，但多批次染色时间应相同，染色强度相同。 </li><li>倒出胭脂红色明矾溶液，并在100％蒸馏水中冲洗腺体30秒。在70％乙醇中浸泡15分钟，95％乙醇浸泡15分钟，逐渐脱水腺体和100％乙醇20分钟。 </li><li>根据厚度，将其浸泡在二甲苯中12-72小时，清除脂肪腺体。 注意:腺体应为半透明（透明）。如果任何不透明（白色）区域保留，继续浸泡在二甲苯直到半透明。如果泌乳或其他非常厚的腺体被染色和清除，则二甲苯可能需要更换一次才能完全清除腺体。 </li><li>通过移液足够的介质只覆盖腺体，用二甲苯安装介质安装载玻片。添加盖玻片，确保不会形成气泡。 </li><li>让幻灯片干燥。当安装介质干燥时，它将在盖玻片下收缩，可能需要添加额外的安装介质。一旦不需要额外的介质，允许滑块完全干燥;这可能需要2-3周。 </li><li>一旦载玻片完全干燥，可以用棉签和少量二甲苯除去任何残留的固定介质。小心不要使用太多的二甲苯，因为这可以溶解安装介质并松开盖玻片。如果发生这种情况并且气泡聚集在盖玻片下面，则盖玻片应该在二甲苯中除去，并且重复安装过程。 </li></ol> 3.准备整体图像进行分析<ol><li>使用宏观显微镜或解剖显微镜和数码相机使用适当的软件捕获整体安装的图像（ 图3 ）。 注意:虽然可以选择捕获整个腺体上皮的任何放大倍数，但是必须捕获将以相同放大倍数彼此进行比较的所有全贴图像。 </li><li>下载ImageJ软件（或FIJI软件） 6 。 </li><li> 通过点击"文件"→"打开"打开ImageJ中的乳腺整体图像。选择手写工具并追踪腺体上皮。选择"编辑"＆＃8594; "外界清晰"。 <ol><li>通过跟踪节点并使用手写工具和"编辑"→"剪切"来删除淋巴结。 </li></ol></li><li>通过选择"图像"→"颜色"→"分割通道"来分离颜色通道。选择具有最佳对比度的通道，通常为蓝色通道。 注意:RGB图像由该图像的红色，绿色和蓝色分量的堆叠组成。此操作将这些组件分为三个8位灰度图像。 </li><li>通过选择"处理"→"减去背景"来减去背景。选择所需的参数，然后单击"预览"以预览更改。 注意:"减去背景"可以消除平滑，连续的背景。另外，可以使用"过程"→"过滤器"→"无刷面罩"来创建对比度。 </li><li> 选择其中之一<html> e自动消除噪音的以下方法:去除斑点或去除异常值。 注意:ImageJ提供了自动消除噪声的第三种方法:去除NaN。但是，删除NaNs命令不适用，因为它使用32位映像，而当前方法使用8位映像。 <ol><li>通过选择"处理"→"噪声"→"去斑点"，使用去斑指令去除噪声。 注意:这相当于添加一个中值滤波器，它将每个像素替换为其3×3邻域中的中值。 </li><li>通过选择"处理"→"噪声"→"删除异常值"，使用"删除离群值"命令消除噪点。 注意:如果偏离中位数超过某个值（阈值），此过程将替换像在周围环境中像素的中值的像素。 </li></ol></li><li> 手动清除任何剩余噪音（lass ="xfig"&gt;图4）。 <ol><li>打开原始图像的副本，并使用它作为指导，什么是什么和什么是不噪音。单击工具栏最右侧的双重红色箭头按钮。选择绘图工具;绘图工具按钮现在将显示在工具栏中。 </li><li>单击橡皮擦工具。通过右键单击橡皮擦工具按钮来调整橡皮擦直径。按住鼠标左键消除噪点。 注意:只有一个删除会话可以撤消。一旦鼠标左键被释放并再次点击，上一次的擦除将无法撤消。 </li></ol></li><li> 通过选择"图像"→"调整"→"阈值"来调整阈值。移动滑块以调整最小（上滑块）和最大（下滑块）阈值，直到达到压盖的适当描述。 注意:设置阈值将灰度图像分段为感兴趣的和背景的特征。<ol><li>单击应用。如有必要，请按照步骤3.6.1和3.6.2删除此处的附加噪声。 </li></ol></li><li> 重建通过阈值和噪声去除去除的腺体上皮的部分（ 图5 ）。 <ol><li>仔细进行图像重建，并保持原始图像的完整性。参考原始图像，以了解什么是什么和什么不是上皮。 </li><li>单击喷雾工具（在工具栏上使用绘图工具）。通过右键单击Spray Can工具按钮调整喷雾直径和速率。通过点击或按住鼠标左键，小心地填入腺体的缺失部分。 </li></ol></li><li> 创建一个用于进行Sh​​oll分析的腺体的骨架图像。通过选择"处理"→"二进制"→"制作B"，确保阈值图像为二进制图像<html> inary"。 <ol><li>如果图像在黑色背景上为白色，请选择"处理"→"二进制"→"选项"，然后取消选中"黑色背景"。通过选择"进程"→"二进制"→"骨骼化"来对图像进行骨骼化。 注意:这将重复从二进制图像的边缘删除像素，直到其缩小为单像素宽的形状。 </li><li>通过选择"过程"→"二进制"→"扩张"来一次填充图像。 注意:这通过将像素添加到二进制图像的边缘来填补由阈值化和骨架化创建的间隙。 </li></ol></li><li>选择"文件"→"另存为"保存图像。选择图像类型（通常为jpeg），输入文件名，然后单击"确定"。 </li><li> 通过将骷髅图像叠加到原始图像上来检查骷髅图像的准确性（ass ="xfig"&gt;图6）。 <ol><li>通过打开原始图像和骨架化图像来创建叠加层。选择"图像"→"覆盖"→"添加图像"。在"添加图像"对话框中，从"图像添加"下拉菜单中选择骨架图像，并将不透明度设置为30％。 </li><li>通过选择"文件"→"另存为"，将覆盖的骨架图像的图像保存到原稿上。选择图像类型，输入文件名，然后单击"确定"。 </li></ol></li></ol> 4.进行Sholl分析<ol><li> 打开骨架图像，并通过选择"进程"→"二进制"→"制作二进制"使图像二进制。 <ol><li>在设置放大倍率之前，使用千分尺测量拍摄图像的放大倍率的像素数/ mm。 </li><li>设置测量的倍率s选择"分析"→"设置比例"。输入像素数/ mm。将"已知距离"和"像素长宽比"设置为1.为"单位长度"输入"mm"，并选中"全局"（每个图像保持相同的比例）。 </li></ol></li><li> 通过从主导管开始（分析中心）到腺体上皮的最远端点绘制一条线，确定Sholl分析的结束半径（包围半径）（ 图7 ）。 <ol><li>使用工具栏中的线条绘图工具在感兴趣的点之间画一条线。按"M"键进行测量，并注意结果窗口将打开。 注意:"长度"列中的值是以mm为单位的行长度。设置Sholl参数时，该值将自动输入为结束半径。 Sholl插件将使用起点的线作为中心环的中心。 </li></ol></li><li> 运行Sholl分析。 <ol><li>通过选择"插件""高级分析"来运行分析;将出现一个参数窗口。 </li><li>将"I.壳体定义"中的起始半径设置为0.00 mm;在步骤4.2中测量的线的长度将自动输入为"结束半径"。 </li><li>将"半径步长"设置为0.1 mm。 注意:半径步长确定环数（有效地迭代次数）;较小的步距将增加环的数量，而较大的步长将减少环数。半径不能太小，虽然半径过小会导致不必要的环数。然而，它可以设置得太大，这将产生较少的环，并且随后低估了交叉点的真实数量。参考Stanko 等（2015），以获取有关确定步长的更多信息。 </li><li>在"II。每个半径多个样本"中将"＃样本"设置为1，将"积分"设置为"均值"。 </li><li>将"封闭半径截止"设置为1，并在"III。描述符和曲线拟合"中选择"起始半径推断"为"＃主分支"。 注意:可以根据需要检查"适合配置文件和计算描述符"和"显示配件详细信息"。 </li><li>检查"线性"，并在"不符合标准化的配置文件"中选择"最佳配合度";检查"最有信息"。在"IV。Sholl方法"中选择"规范化配置文件的区域"。 </li><li>在"V.输出选项"中选中"创建交点掩码"，以创建交点的热图（可选）。 </li><li>单击"Cf. Segmentation"生成具有振铃的图像的预览窗口以确认该区域的分析。 </li></ol></li><li>单击"确定"运行分析。 </li></ol> 5.测量MEA <ol><li> 通过追踪上皮树周围的最短距离来测量MEA（ 图8 ）。 <ol><li>在ImageJ中，打开压盖的骨架图像，单击多边形工具。点击上皮树周边的一个点，开始多边形，围绕腺体的周边移动，然后单击以添加一个线段。 </li><li>当整个上皮区域被绕过时，双击关闭多边形。按"M"键打开结果窗口; "区域"列中的值是MEA。 </li></ol></li><li> 在腺体上皮超过淋巴结的情况下，在计算分支密度时，从MEA中减去淋巴结面积（LNA）。 <ol><li>通过跟踪同一个人的淋巴结来测量LNA呃为上皮树，按"M" 注意:在这些情况下，必须从MEA中减去LNA，因为淋巴结阻止分析计算LNA内的交点。当上皮未达到淋巴结时，LNA为零。 </li></ol></li></ol>报告数据<ol><li> 报告包围半径MEA，N， k和分支密度的值。 注意:在步骤4.2中确定包围半径，并在步骤5.1中确定MEA。 <ol><li>运行分析生成一个Sholl结果窗口。 注意:报告的N是和相的值。报告的k是Sholl回归系数（半对数）的值。 Sholl分析将在腺体上皮的任何测量区域上返回k值。为了在整个上皮上获得k的准确值，必须存在导管末端。 </li><li>修改th通过使用N的输出值计算分支密度（该方法的基本终点），对乳腺进行分析。使用公式N /（MEA-LNA）计算分支密度，并将该值报告为N / mm 2 。 </li></ol></li></ol>

<ol>
	<li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+of+the+neurons+in+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization of the neurons in the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Ferreira, T. A., Iacono, L. L., Gross, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+receptor+1A+modulates+actin+dynamics+and+restricts+dendritic+growth+in+hippocampal+neurons.">Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons.</a> Eur J Neurosci. 32 (1), 18-26 (2010).</li><li>Stanko, J. P., Easterling, M. R., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+Sholl+analysis+to+quantify+changes+in+growth+and+development+in+rat+mammary+gland+whole+mounts.">Application of Sholl analysis to quantify changes in growth and development in rat mammary gland whole mounts.</a> Reprod Toxicol. 54, 129-135 (2015).</li><li>Assis, S., Warri, A., Cruz, M. I., Hilakivi-Clarke, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+Mammary+Gland+Morphology+and+Breast+Cancer+Risk+in+Rats.">Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats.</a> J. Vis. Exp. (44), e2260 (2010).</li><li>Plante, I., Stewart, M. K., Laird, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Mammary+Gland+Development+and+Function+in+Mouse+Models.">Evaluation of Mammary Gland Development and Function in Mouse Models.</a> J. Vis. Exp. (53), e2828 (2011).</li><li>ImageJ User Guide. IJ 1.46r. ImageJ Available from: <a target="_blank" href="https://imagej.nih.gov/ij/docs/guide/146.html">https://imagej.nih.gov/ij/docs/guide/146.html</a> (2012)</li><li>Tucker, D. K., Foley, J. F., Hayes-Bouknight, S. A., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+High-quality+Hematoxylin+and+Eosin-stained+Sections+from+Rodent+Mammary+Gland+Whole+Mounts+for+Histopathologic+Review.">Preparation of High-quality Hematoxylin and Eosin-stained Sections from Rodent Mammary Gland Whole Mounts for Histopathologic Review.</a> Toxicol Pathol. 44 (7), 1059-1064 (2016).</li><li>Fenton, S. E., Hamm, J. T., Birnbaum, L. S., Youngblood, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+abnormalities+in+the+rat+mammary+gland+following+gestational+and+lactational+exposure+to+2,3,7,8-tetrachlorodibenzo-p-dioxin+(TCDD).">Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).</a> Toxicol Sci. 67 (1), 63-74 (2002).</li><li>Murray, T. J., Maffini, M. V., Ucci, A. A., Sonnenschein, C., Soto, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammary+gland+ductal+hyperplasias+and+carcinoma+in+situ+following+fetal+bisphenol+A+exposure.">Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure.</a> Reprod Toxicol. 23 (3), 383-390 (2007).</li><li>Moral, R., Santucci-Pereira, J., Wang, R., Russo, I. H., Lamartiniere, C. A., Russo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+exposure+to+butyl+benzyl+phthalate+induces+modifications+in+the+morphology+and+the+gene+expression+profile+of+the+mammary+gland:+an+experimental+study+in+rats.">In utero exposure to butyl benzyl phthalate induces modifications in the morphology and the gene expression profile of the mammary gland: an experimental study in rats.</a> Environ Health. 10 (1), 5 (2011).</li><li>Johnson, M. D., Mueller, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+dimensional+multiphoton+imaging+of+fresh+and+whole+mount+developing+mouse+mammary+glands.">Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.</a> BMC Cancer. 13, 373 (2013).</li><li>Enoch, R. R., Stanko, J. P., Greiner, S. N., Youngblood, G. L., Rayner, J. L., Fenton, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development+as+a+sensitive+end+point+after+acute+prenatal+exposure+to+an+atrazine+metabolite+mixture+in+female+Long-Evans+rats.">Mammary gland development as a sensitive end point after acute prenatal exposure to an atrazine metabolite mixture in female Long-Evans rats.</a> Environ Health Persp. 115 (4), 541-547 (2007).</li><li>Hovey, R. C., Coder, P. S., Wolf, J. C., Sielken, R. L., Tisdel, M. O., Breckenridge, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+mammary+gland+development+in+female+Long+Evans+rats+following+in+utero+exposure+to+atrazine.">Quantitative assessment of mammary gland development in female Long Evans rats following in utero exposure to atrazine.</a> Toxicol Sci. 119 (2), 380-390 (2011).</li><li>Mandrup, K. R., Hass, U., Christiansen, S., Boberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perinatal+ethinyl+oestradiol+alters+mammary+gland+development+in+male+and+female+Wistar+rats.">Perinatal ethinyl oestradiol alters mammary gland development in male and female Wistar rats.</a> Inter J of Androl. 35 (3), 385-396 (2012).</li></ol>

作者没有什么可以披露的。

从出生到青春期，乳腺生长是异速生长的。青春期后，乳腺通过广泛的导管分支和伸长发展，直到乳腺上皮占据整个脂肪垫。分支特征是乳腺发育的一个重要方面，客观量化这些特征的能力对于评估正常乳腺发育和识别早期暴露于乳腺有毒物质的异常发育是非常有用的。 评估形态特征，量化基本尺寸测量和计数乳腺结构是评估乳腺发育的典型方法。然而，由于啮齿动物乳腺?...

乳腺分支是通常被评估为腺体发育指标的特征，但难以客观量化。 1953年，Sholl 1描述了一种用于测量猫的视觉和运动皮质中的神经元树突状结构的方法，Ferriera 等人 2开发了一种用于该技术的插件。因为神经元和乳腺都具有类似的树状结构，所以该插件用于定量乳腺上皮分支密度在青春期大鼠乳腺的2D图像中。选择年龄阶段进行分析，因为断奶是在学术实验室和测试指南研究中经常评估的生命阶段。 Sholl分析是与FIJI一起分发的插件，它是开源图像处理软件包ImageJ，并附加了一些插件。该插件创建了一系列围绕predef的同心环（通常是神经元的神经元或乳腺的主要管道的起源）并延伸到物体的最远部分（包围半径）。然后计算每个环上发生的交点数（N）。该插件还返回一个Sholl回归系数（ k ），它是上皮分支衰减速率的量度。 使用ImageJ，创建乳腺整体的骨架图像，并测量乳腺上皮区域（MEA）。使用Sholl分析插件分析图像，并返回N和k的值以及其中未使用的其他值。通过计算N / MEA确定乳腺上皮分支密度。分支持续在腺上皮的外部区域的程度是分支复杂性，并且是均匀的远端上皮生长的指标。因为k是海拔远端下降的量度elial分支，它是分支复杂性的有效措施和乳腺发育的可靠指标。 该协议描述了一种计算机辅助方法，用于创建乳腺整体的骨架图像，并定量评估年龄男性和雌性大鼠的乳腺分支特征。这种方法相对较快，不需要使用专门的显微镜设备。该方法的开发和验证在Stanko 等人 （2015） 3 。本报告还介绍了大鼠乳腺整体的制备方法。 de Assis 等人已经描述了类似的乳腺整体手术（2010） 4和Plante et al。 （2011） 5 。

<table border="0" cellpadding="0" cellspacing="0" width="1069">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Dissecting board</td>
			<td style="width:161px;">NA</td>
			<td style="width:224px;">NA</td>
			<td style="width:439px;">A piece of styrofoam roughly 10&#34;x12&#34; is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Dissecting T-Pins</td>
			<td>Daigger</td>
			<td style="width:224px;">EF7419A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Spray bottle with ethanol</td>
			<td>NA</td>
			<td style="width:224px;">NA</td>
			<td>70% ethanol is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Curved dissecting scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14569-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Straight dissecing scissors</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">14568-09</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Curved forceps</td>
			<td>Fine Science Tools</td>
			<td style="width:224px;">11003-12</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:245px;">Superfrost Plus 24x75x1 mm microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>4951PLUS-001</td>
			<td style="width:439px;">Thermo Scientific Superfrost Plus &#38; Colorfrost Plus slides hold tissue sections on permanently without the need for expensive coatings in IHC and Anatomical Pathology applications. This treatment reduces tissue loss during staining as well as hours of slide preparation. Slides electro-statically attract frozen tissue sections and cytology preparations and feature a chemistry similar to silane, although optimized to improve application performance. 
			https://www.thermofisher.com/order/catalog/product/4951PLUS4.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Fisherfinest Premium Cover Glass 24x60x1 mm</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">12-548-5P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">Bemis Parafilm M Laboratory Wrapping Film</td>
			<td>Fisher scientific</td>
			<td style="width:224px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>C2432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>A9967</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Ethanol absolute, &#8805;99.8% (GC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:224px;">24102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carmine alum&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum potassium sulfate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Permount mounting media</td>
			<td>Fisher Scientific</td>
			<td style="width:224px;">SP15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Macroscope</td>
			<td>Leica</td>
			<td>Z16 APO&#160;</td>
			<td rowspan="3" style="width:439px;">This is the image capturing hardware and software used in this laboratory.&#160; As there are many different options, the methods and applications may vary between laboratories.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Digital camera</td>
			<td>Leica</td>
			<td style="width:224px;">DFC295</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Camera software</td>
			<td>Leica</td>
			<td style="width:224px;">Leica Application Suite v3.1&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">ImageJ software</td>
			<td>Open source</td>
			<td>http://imagej.net/Welcome</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Sholl analysis&#160;</td>
			<td>Open source</td>
			<td>http://imagej.net/Sholl_Analysis</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying branching density in rat mammary gland whole-mounts using the sholl analysis method

越来越多的研究正在利用啮齿动物乳腺作为评估化学物质暴露的发育毒性的终点。这些暴露对乳腺发育的影响通常使用基本尺寸测量或通过评定形态学特征来评估。然而，解释发展变化的广泛方法可能导致实验室之间的翻译不一致。需要一种常见的评估方法，以便从研究中进行比较的数据形成适当的解释。本研究描述了Sholl分析方法应用于定量乳腺分支特征。 Sholl方法最初开发用于量化神经元树突状模式。通过使用ImageJ，开源图像分析软件包和为此分析开发的插件，乳腺分支密度和复杂性确定来自年龄大的雌性大鼠的乳腺。这里描述的方法将能够使用Sholl分析作为定量乳腺发育重要特征的有效工具。

在本方案中分析的乳腺的测量封闭半径，MEA，N， k和计算的分支密度的值报告在表1中 。 Sholl分析产生每个半径处的交点数（ 图9 ）的线性和半对数图 ，如果选择，则生成交点的热图（ 图10 ）。欠发达的腺体在同一MEA内表现出较少的交叉点，因此具有较低的支化密度。发育良好的腺体?...

Watch this Scientific Journal Video about Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method at JoVE.com

Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

通常使用描述性评估或通过测量基本物理属性来评估啮齿动物中的乳腺发育。分支密度是乳腺发育的一个指标，难以客观量化。该方案描述了乳腺分支特征定量评估的可靠方法。

使用Sholl分析方法量化大鼠乳腺组织中的分支密度

An increasing number of studies are utilizing the rodent mammary gland as an endpoint for assessing the developmental toxicity of a chemical exposure. The ...

quantifying-branching-density-rat-mammary-gland-whole-mounts-using

Research

JoVE Journal

Developmental Biology

14.1K 次观看.  National Institute of Environmental Health Sciences. 通常使用描述性评估或通过测量基本物理属性来评估啮齿动物中的乳腺发育。分支密度是乳腺发育的一个指标，难以客观量化。该方案描述了乳腺分支特征定量评估的可靠方法。 

视频: Quantifying Branching Density in Rat Mammary Gland Whole-mounts Using the Sholl Analysis Method

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.

通过细胞外囊泡/外来体转移乳腺基质上皮细胞和乳腺腔细胞之间的乳腺形成能力

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

新型乳腺脂肪移植技术可视化血管内皮干细胞的血管生成

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

一种对绿色藻原体同步文化进行深入分析的多表率提取方法

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...