这项工作得到了 SBIR 赠款 (1R44EY027654-01A1) 的支持。

所有与老鼠有关的程序都是按照儿童医院奥克兰研究所动物保育和使用委员会批准的协议进行的, 并加入了视觉和眼科研究协会 (ARVO) 的声明, 供使用动物的眼科和视力研究。
1. 染色的组织准备
<ol><li>弄死 C57 BL/6J 鼠胚胎日 (E) 16, 产后日 (P) 0, P15 和 P90 的二氧化碳 (CO2) 后, 斩首 (E16 和 P0 小鼠)。</li>
	<li>立即 enucleate 鼠眼刺穿与 29G1/2 针在背侧的眼睛。在1毫升4% 多聚甲醛 (粉煤灰) 中孵育5分钟 (最小), 磷酸盐缓冲盐水 (1x PBS) 在室温下 pH 为8.0。</li>
	<li>从 P0、P15 和 P90 制作眼杯, 用细眼剪刀解剖角膜和晶状体, 而眼睛在4% 的粉煤灰中。 注: 跳过这一步为 E16 眼睛, 因为眼睛太小。</li>
	<li>将眼杯 (P0, P15 和 P90) 和整个眼睛 (E16) 固定在4% 的粉煤灰中, 室温20分钟。然后在1毫升 1x PBS 中洗两次眼杯和全眼睛, 室温10分钟。</li>
	<li>Cryoprotect 眼杯和全眼睛在 1xPBS, pH 8.0 含10% 蔗糖1小时。保持20% 蔗糖在 1x pbs 1 小时, 然后饱和30% 蔗糖在 1x PBS 隔夜在4摄氏度。</li>
	<li>第二天, 将眼杯和整个眼睛嵌入到最佳切削温度化合物 (OCT) 中, (500 µL) 在 cryomolds 中, 在干冰/乙醇浴中凝固, 并贮存在-80 摄氏度。</li>
	<li>删除模具与嵌入式眼杯和整个眼睛从-80 °c, 并允许平衡到-20 °c 1 小时前 cryosectioning。</li>
	<li>将视网膜横断面 (12 µm 厚) 与 temporonasal 轴平行, 通过视神经头使用恒温器在-20 摄氏度。</li>
	<li>在显微镜上安装视网膜切片53和存储在-80 摄氏度。</li>
</ol>2. 与 5-mC 和5染色抗体的结合
<ol><li>用疏水性屏障笔将安装在幻灯片上的视网膜部分包围起来。疏水性屏障笔减少染色组织所需的抗体量。</li>
	<li>用200µL 1x PBS 冲洗视网膜部分10分钟。</li>
	<li>在室温下, 在 1x pbs (pbs t) 中, Permeabilize 200 µL 0.1% 的海卫 X-100 的视网膜部分, 10 分钟。</li>
	<li>变性视网膜部分为30分钟与200µL 新鲜地做2盐酸 (HCl) 酸在 1x PBS 在37°c 孵化器。 注意: 需要对 HCl 进行仔细的滴定, 以优化5mC 信号。</li>
	<li>在优化5mC 信号54时, 始终包括生物复制 (3-4)。 注: 我们在4个时间点 (15 分钟, 30 分钟, 1 小时和2小时) 进行了抗原检索, 发现30分钟孵化是最佳的5mC 信号。用 HCl 进行长时间的孵化会降低细胞核结构, 导致无法将5mC 信号定位到细胞核。</li>
	<li>变性后, 在视网膜部分增加100µL 0.1 米 (pH 8.3), 以10分钟内中和视网膜部分。</li>
	<li>在500µL 的阻塞溶液 (5% preimmune 正常山羊血清0.1% 海卫 X-100 在 1x PBS) 孵化的部分, 在室温下的湿度室1小时。</li>
	<li>阻塞后, 添加 anti-5mC;anti-5hmC 抗体稀释1:500 在阻断溶液的视网膜部分。</li>
	<li>在湿度室中过夜4摄氏度的视网膜部分孵化。</li>
	<li>第二天洗涤, 视网膜部分3次 (每次10-15 分钟) 与 0.1% PBS T, 然后孵化在阻断溶液与相应的二级抗体 (山羊抗兔和山羊抗鼠 IgG, 稀释; 1:1000), 含 DAPI (4 ', 6-diamidino-2-phenylindole) 溶液 (1 µg/毫升) 在室温下为1小时的阻塞溶液。 注意: 避免在 immunodetection 的所有阶段干燥部分。</li>
	<li>用1毫升 0.1% PBS 清洗幻灯片三次。</li>
	<li>最后一次清洗后, 在盖玻片下装上带有水的安装介质的部分, 用无色指甲油密封。</li>
</ol>3. 共聚焦免疫组化
<ol><li>将切片定向到光学杯的视网膜色素上皮侧总是面对一种方式 (例如,向上), 以保持一致性。</li>
	<li>以2X 或3X 变焦的 63X (物镜放大倍数) 对 immunostained 视网膜切片进行图像采集。</li>
	<li>使用0.35 µm 步骤, 在所有3通道 (红色、绿色和蓝色、RGB) 中生成每个选定图像 (z栈) 的多个光学部分。 注: 在 10-20X (物镜放大倍数) 的样品的最终放大倍数将分别为 63X, 当与 (通常使用的) 10X 眼透镜结合时。 可视化压缩光栈定位5mC 和5hmC 信号</li>
</ol>

作者没有什么可透露的。

dna 甲基化和羟甲基化是动态和可逆的 dna 修饰, 它 modulatethe 在一个发展中和分裂后细胞中的不同范围的生物机制。在这里, 我们描述了一种可重现的技术, 通过免疫组化技术 (使用 anti-5mC 和 anti-5hmC 抗体) 在多聚甲醛固定的小鼠视网膜冷冻切片中检测原位的5mC 和 5hmC DNA 修饰, 并提供改进和标准化结果的准则, 特别是在比较几种不同的标本时。我们早先使用这种方法描绘了小鼠视网膜的5mC 变化与视网膜特异靶向Dnmt1, Dnmt3a和Dnmt3b DNA 甲基基因, 并报告了 (预期) 耗尽5mC 标记在他们的视网膜7.
5mC 免疫组化检测的关键步骤是优化用 HCl 治疗组织学切片: 不到15分钟的预处理不会产生可重现的结果, 而 HCl 的过度治疗会破坏核建筑。在30分钟的潜伏期后, 我们发现了最好的结果。我们建议始终使用新鲜稀释盐酸和新鲜的4% 粉煤灰溶液的 DNA 变性和视网膜组织固定。
为了解决这些结果, 我们建议在优化5mC 信号时包括三到四个生物复制。虽然每一组免疫组化染色可能产生略有不同的结果 (或多或少明亮的异色区域), 这是预期的免疫组织化学方法, 5mC 和5hmC 核分布在单独的集合由于遵循了该协议, 预计各节的可重现性非常好。
这一技术也有局限性, 因为我们一贯观察的变化, 在5mC 分布在感光细胞细胞核内的同一部分。我们发现一些细胞核显示强的5mC 信号, 而相邻的细胞核显示没有5mC 信号。这是由于在冷冻切片中盐酸处理揭露5mC 抗原的限制。
这一技术的意义, 使5mC 定位没有 DNase 和蛋白酶 K 治疗, 从而更好地保存 chromocenters。这项技术有望在所有脊椎动物组织的任何部分, 携带 5mC, 5hmC 标记, 并处理与类似的方法, 不仅是老鼠视网膜, 只要遵循该协议。

小鼠视网膜发育和分裂后稳态的表观遗传调控是一个令人兴奋的研究领域, 它有望带来对控制 retinogenesis 和细胞命运确定的生物学机制的新理解, 细胞类型特异性新陈代谢的作用并且细胞病症, 细胞死亡并且再生1,2,3,4,5,6,7,8,9,10,11,12,13. 染色质经历了发展的动态变化, 以及对可变外部信号的反应, 无论是压力、新陈代谢还是细胞死亡刺激6、14、15,16,17,18. 在小鼠细胞核中, 染色质分为活性染色质和非活动染色质6、19、20。在相间的细胞核中, 染色质位于原子核的内区, 而染色质则系核外围和核仁。这种模式被称为常规, 是高度保守的真核生物。相比之下, 夜间动物中的棒核, 如老鼠和大鼠, 有颠倒的模式, 其中原子核被染色质所包围的中心被染色质形成最外层的外壳6,18, 20。在出生时, 棒核具有传统的核结构。通过对常规核结构的重塑, 在发展过程中出现了杆核的反演。在这个过程中, 外围染色质分离从核外围和 chromocenters 保险丝一起形成一个单一的 chromocenter 在核心6,18。
基因组 DNA 甲基化参与控制局部染色质构象21。DNA 甲基化发生在 dinucleotides 的胞嘧啶残留物的 5 ' 位置上, 在小鼠基因组22、23、24、25、26中的许多基因的启动子区域富集。并且在基因间和内含子区域, 运载调控元素27。21、24和 cpg 基因促动剂27、28中 cpg 岛的甲基化是调节二价染色质动态状态的重要因素, 含有许多发育基因/转录因子在发育、癌症和衰老中扮演重要角色29、30、31、32、33、34.三 dna 甲基转移酶 (DNMTs) 参与小鼠发育过程中的 dna 甲基化35。所有三 DNMTs 都表达在小鼠视网膜发育36和视网膜特异的变化Dnmt基因影响视网膜发育2,7。Hydroxymethylcytosine (5hmC) 是 5-甲基胞嘧啶 (5mC) 去甲基化的氧化产物。三十-十一易位 (春节) 酶参与5mC 去甲基化37,38,39。在基因丰富和活跃转录区27、40中, 促进剂、基因体和基因间基因组序列丰富了羟甲基 C 的修饰。
在41、42、43、44、45、46等细胞中, 有多种方法来确定 DNA 甲基化模式的变化, 其中一些能使对 DNA 甲基化的全球变化进行量化, 而另一些则侧重于促进者和促进者中富中央地区的精确变化。还报告了5hmC 的检测和定量方法,包括免疫组化13。免疫组化技术能够对发育和分裂后细胞细胞核内的5mC 和5hmC 变化进行强健的监测, 对于根据外部或内部变化在原位划定染色质动力学非常有用。影响细胞4,13,48,49。然而, 这种方法容易低估 DNA 甲基化和羟甲基化变化由于技术上的困难, 与检测胞嘧啶修改的组织学准备。这是因为非极性 dna 基地, 包括5mC 和 5 hmc 修饰胞嘧啶, 隐藏在双链 DNA 螺旋线的中心50, 并要求揭露。这是特别具有挑战性的冷冻组织学准备, 这可能会迅速失去信息组织的原始组织时, 苛刻的治疗应用。
小鼠视网膜是解剖 DNA 甲基化对神经发育和分裂后稳态的贡献的优秀模型。只有6种类型的神经元 (棒和锥感光细胞, 无长突, 双极, 水平和神经节的单元), 一个胶质细胞类型 (穆勒胶质) 和一个神经上皮细胞类型 (视网膜色素上皮)51。据报道, 视网膜细胞类型有不同的 DNA 甲基化18,19的模式, 最近已经研究了在单基分辨率1,5,9,12。我们最近报告了免疫组化应用, 以划定5mC 分布模式的视网膜细胞核内和成年小鼠视网膜细胞核的变化, 其中所有三 DNA 甲基转移酶已被删除的条件靶向7. 与一些报告相比, 在视网膜细胞中 DNA 甲基化的存在只能看作是 hypermethylated 细胞凋亡的阳性或阴性信号, 我们的方法能够检测到特定的染色质安排在视网膜细胞核之内, 我们和几个小组报告和讨论了早先5,6,7,18,19,36,52. 在这里, 我们描述了免疫组化 (IHC) 技术检测5mC 和5hmC 在冷冻多聚甲醛固定组织学切片的细节, 并证明了数据, 我们能够从我们的原始报告复制7.

<table><tbody><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>29G1/2 ULTRA-FINE</td><td>BD Biosciences</td><td>329461</td><td></td></tr><tr><td>Ophthalmic scissor</td><td>Fine Science tools, Inc.&amp;nbsp;</td><td>15000-03</td><td></td></tr><tr><td>PAP pen</td><td>RPICORP.com</td><td>195505</td><td></td></tr><tr><td>Microslide Superfrost plus</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscope Science</td><td>15710</td><td></td></tr><tr><td>1x PBS</td><td>Corning</td><td>21-031-CV</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>Tissue-Tek Optical cutting compound (OCT)</td><td>VWR</td><td>4583</td><td></td></tr><tr><td>Triton X 100</td><td>Sigma</td><td>T9284</td><td></td></tr><tr><td>6 N HCl</td><td>VWR analytical&amp;nbsp;</td><td>BDH7204-1</td><td></td></tr><tr><td>Tris-HCl pH 8.3</td><td>Teknova</td><td>T1083</td><td></td></tr><tr><td>Goat serum</td><td>Jackson Immunoresearch</td><td>005-000-121</td><td></td></tr><tr><td>5mC</td><td>Genway Biotech&amp;nbsp;</td><td>GWB-BD5190</td><td></td></tr><tr><td>5hmC</td><td>Active Motif</td><td>39791</td><td></td></tr><tr><td>LaminB1</td><td>Abcam</td><td>Ab16048</td><td></td></tr><tr><td>Cy2 AffiniPure Goat Ant-Rabbit IgG</td><td>Jackson Immunoresearch</td><td>111-225-1444</td><td></td></tr><tr><td>Cy3 AffiniPure Goat Ant-Mouse IgG</td><td>Jackson Immunoresearch</td><td>115-585-166</td><td></td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Prolong Gold Antifade medium</td><td>Thermo Fisher Scientific</td><td>P36930</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MICRM HM 550</td><td>Thermo Fisher Scientific</td><td>388114</td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

immunohistochemical detection of 5-methylcytosine and 5-hydroxymethylcytosine in developing and postmitotic mouse retina

视网膜发育的表观遗传学是一个很好的研究领域, 它有望为人类视网膜退行性疾病的机制带来新的认识, 并找出新的治疗方法。小鼠视网膜的核结构有两种不同的模式: 常规和倒置。传统的模式是普遍的地方, 染色质是局部的核心, 而活跃的染色质驻留在核内部。相反, 反转核模式是唯一的成人棒感光细胞细胞核, 染色质本地化到核中心, 染色质驻留在核外围。chromocenters 中主要观察到 DNA 甲基化。DNA 甲基化是在许多基因的启动子区域丰富的 dinucleotides 胞嘧啶残留物 (5-甲基胞嘧啶, 5mC) 的动态共价修饰。三 dna 甲基转移酶 (DNMT1、DNMT3A 和 DNMT3B) 在发育过程中参与 dna 甲基化。用免疫组化技术检测5mC 是非常有挑战性的, 导致结果的变异性, 因为所有的 dna 基地, 包括5mC 修改的基地都隐藏在双链 dna 螺旋。然而, 在开发过程中详细划定5mC 分布是非常有用的。在这里, 我们描述了一种可重现的技术, 用于健壮的免疫组化检测5mC 和另一种表观遗传 DNA 标记 5-hydroxymethylcytosine (5hmC), colocalizes 与 "开放", 转录活性染色质在开发和分裂后老鼠视网膜。

为确定 5-甲基胞嘧啶 (5mC) 和 5-hydroxymethylcytosine (5hmC) 在视网膜发育和分裂后视网膜中的分布, 我们 immunostained C57BL/6J、E16、P0 和 P15 的小鼠视网膜切片, 其抗体特定于5mC 和5hmC。
在 E16, 5mC 染色在细胞核的 chromocenters 中很强, 而在整个细胞核中观察到微弱的染色 (图 1a)。5hmC 染色仅限于细胞核, 未从 chromocenters (图 1b) 中消失。
在 P0 视网膜中, 5mC 信号被定位到细胞核和核外围的 chromocenters (图 2a、箭头和箭头)。我们还观察到细胞细胞核 chromocenters 的弱染色5mC。除了 chromocenters (图 2b) 外, 5hmC 信号在细胞核中强烈存在。我们发现在内 neuroblast 层 (INBL) 中有更多的细胞核被染色5mC 和 5hmC (图 2c, 虚线)
在 P15 视网膜, 我们发现在外层核层 (一), 内核层 (INL) 和神经节细胞层 (图 3) 强染色5mC 和5hmC。在 chromocenters (棒感光细胞) 中, 5mC 信号存在于细胞核和核外围 (图 3 a-3 c, 3g) 中。除了 chromocenters (图3维-3 f) 外, 整个细胞核中都发现了5hmC 信号。在 P15 视网膜上进行的透射电镜显示异色域在棒状细胞核中的分布, 类似于5mC 抗体信号 (图 3h和3i)。
INL 和协鑫的5mC 染色在细胞核的 chromocenters 中很强, 在整个细胞核中也观察到弱染色 (图 3 j-3 升和3 r-3 t)。与5mC 相比, 5hmC 信号在 INL 和协鑫 (图 3 m-3 o和3 u-3 w) 的 chromocenters 中被本地化为整个细胞核。我们在协进细胞细胞核的外围观察到强的5hmC 信号。
在成人棒感光细胞中, 5mC 信号被限制在细胞核的 chromocenter 和核外围 (图 4 a-4 c), 而5hmc 信号仅限于核外围 (图 4d-4f)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58274/58274fig1.jpg"> 图1。5-甲基胞嘧啶和 5-hydroxymethylcytosine 在小鼠视网膜胚胎日的定位16。 染色 C57BL/6J 视网膜的抗体 5mC (红色, a) 和 5hmC (绿色, b)。在 E16, 5mC 信号在细胞细胞核的 chromocenters 中非常强, 并且在整个细胞细胞核中也存在很低的水平。5hmC 染色仅限于细胞核。面板 c 表示合并的5mC 和5hmC 图像。细胞核与 DAPI (蓝色, c) 复染。嵌入表示图像中显示星号的区域的放大倍数。刻度条: 5 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58274/58274fig1large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58274/58274fig2.jpg"> 图2。5-甲基胞嘧啶和 5-hydroxymethylcytosine 在小鼠视网膜上的免疫组化染色0。 Immunolabeling C57BL/6J 视网膜的抗体 5mC (红色, a) 和 5hmC (绿色, b)。在 P0 视网膜, 5mC 和5hmC 均存在于外 neuroblast 层 (ONBL) 和内 neuroblast 层 (INBL)。a) 5mC 信号强烈存在于细胞核 (箭头) 的 chromocenters 和核外围。细胞细胞核的 chromocenters 也观察到5mC 的弱染色。b) 5hmC 染色仅限于细胞核, 在 INBL (c 面板的虚线) 中观察到强信号。面板 c 表示合并的5mC 和5hmC 图像。细胞核与 DAPI (蓝色, c) 复染。嵌入表示图像中显示星号的区域的放大倍数。刻度条: 5 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58274/58274fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58274/58274fig3.jpg"> 图3。5-甲基胞嘧啶和 5-hydroxymethylcytosine 分布在小鼠视网膜在出生日15。 染色 C57BL/6J 视网膜的 anti-5mC 和5hmC 抗体 (a-g, j y)。在外层核层 (chromocenters) 中, 5mC 信号 (红色, a) 通常与细胞细胞核的 (箭头, b) 相重合, 也本地化到核外围 (箭头, b)。面板 b (红色) 和 c (灰色) 表示在图像 a 中以星号显示的区域的放大倍数。除 chromocenters 外, 5hmC 信号 (绿色、d) 仅限于整个细胞核。面板 e (绿色) 和 f (灰色) 表示图像 d 中以星号显示的区域的放大倍数. 面板 g 表示合并的5mC 和5hmC 图像。细胞核与 DAPI 复染。面板 h 和 i 是透射电子显微镜图像的感光细胞核在出生日15显示的分布异色领域。面板 h 中的黑色箭头指向面板 i 中放大的单杆感光细胞核. 面板中的红色箭头指向异色在棒状感光核内的域, 而红色箭头指向核外围的染色质。在 INL (j q) 和协进细胞核 (r y), 5mC 信号 (红色) 是本地化的 chromocenters 目前在外围和弱染色也检测在其余的细胞核。面板 k (红色) 和 i (灰色) 表示图像 j 中以星号显示的区域的放大倍数。INL 和协进细胞核中的5hmC 染色 (绿色) 仅限于整个细胞核。注意在协进细胞的核外围有强的5hmC 染色。面板 p 和 x 表示合并后的5mC 和5hmC 图像, 而面板 q 和 y 表示图像 p 和 x 中显示的星号区域的放大倍数。细胞核与 DAPI 复染。刻度条: 5 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58274/58274fig3large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58274/58274fig4.jpg"> 图4。5-甲基胞嘧啶和 5-hydroxymethylcytosine 分布在 C57BL/6J 鼠视网膜棒状感光细胞核90。 5mC 染色 (红色, a) 强烈存在于细胞原子核的 chromocenter 并且微弱地存在在核外围。面板 b (红色) 和 c (灰色) 表示在图像 a 中以星号显示的区域的放大倍数。5hmC 染色 (绿色, d) 仅限于核外围面板 e (绿色) 和 f (灰色) 表示图像 d 中星号显示的面积放大. 面板 g 表示合并的5mC 和5hmC 图像, 而面板 h 是放大的面积显示与图像 g 核中的星号与 DAPI (蓝色) 复染。刻度条: 5 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58274/58274fig4large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

Watch this Scientific Journal Video about Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina at JoVE.com

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

本报告的目的是描述在开发和分裂后小鼠视网膜时, 对表观遗传标记、5-甲基胞嘧啶 (5mC) 和 5-hydroxymethylcytosine (5hmC) 的鲁棒免疫组化检测的协议。

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

immunohistochemical-detection-5-methylcytosine-5

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.9K 次观看.  BioTime, Inc. 本报告的目的是描述在开发和分裂后小鼠视网膜时, 对表观遗传标记、5-甲基胞嘧啶 (5mC) 和 5-hydroxymethylcytosine (5hmC) 的鲁棒免疫组化检测的协议。

视频: Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

科研

遗传学

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

BALB / C脑组织中的高灵敏度检测5 - hydroxymethylcytosine

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

神经科学

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

快速有效地监测完整斑马鱼胚胎中正常和异常胞嘧啶甲基化的时空监测

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

发育生物学

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

生物学

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

小鼠神经干细胞和脑中5-羟基甲基细胞氨酸的检测

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

小鼠视网膜的冷冻切片和免疫染色

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Start this procedure by denaturing the isolated DNA in 0.1 molar sodium hydroxide for 10 minutes at 95 degrees Celsius. Neutralize the DNA with 1 molar ammonium acetate on ice, and then dilute two-fold with double-distilled water.
The most critical step in the dot blot is the spotting of the samples onto the membrane, do it slowly, and carefully. Try to minimize each spotted area to 3 to 4 millimeters in diameter.
Using a narrow-mouth pipette tip, carefully spot 2 microliters of the serial diluted genomic DNA onto a positively-charged nylon membrane at the center of the grid. Blot the membrane at 80 degrees Celsius for 30 minutes. Next, block the non-specific antibody binding sites by soaking the membrane in 5% BSA in TBST in a 10-centimeter Petri dish for 1 hour at room temperature with gentle shaking.
After 1 hour, wash the membrane three times in TBST for 5 minutes each time. Incubate the membrane with a mouse anti-5-methylcytosine monoclonal antibody in TBST at 4 degrees Celsius overnight. On the following day, wash the membrane three times in TBST for 5 minutes each time.
Then, incubate with a secondary antibody &#8212; horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G in TBST for one hour at room temperature. After washing the membrane in TBST as before, add the enzyme substrate to the membrane, and incubate for 5 to 10 minutes. Finally, visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer&#39;s instructions.

5-甲基胞嘧啶和 5-Hydroxymethylcytosine 在小鼠视网膜发育和分裂后中的免疫组化检测

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