这项工作是由奖项资助的。OCE-1323652 通过国家科学基金会海洋科学博士后奖学金和1012629号奖, 来自宝来康威基金博士后浓缩计划。

1. 石蜡的去除
注意: 在通风罩下执行以下步骤。
<ol><li>脱蜡100% 二甲苯下的薄切片石蜡嵌入的幻灯片在玻璃 Coplin 罐子里10分钟。不要使用塑料 Coplin 罐子, 因为二甲苯熔化塑料。使用前, 在高压釜中消毒 Coplin 罐。</li>
	<li>准备四无菌玻璃 Coplin 罐与以下: 100% 乙醇, 80% 乙醇, 70% 乙醇和60% 乙醇。用无 RNase 的水稀释乙醇。<ol>
<li>将幻灯片转移到无菌玻璃 Coplin 罐中, 用100% 乙醇浸泡10分钟。10分钟后, 清空玻璃 Coplin 罐, 替换为新的100% 乙醇。再次浸泡10分钟的幻灯片。</li>
		<li>将幻灯片转移到无菌玻璃 Coplin 罐中, 80% 乙醇为1分钟。</li>
		<li>将幻灯片转移到无菌玻璃 Coplin 罐中, 70% 乙醇为1分钟。</li>
		<li>将幻灯片转移到无菌玻璃 Coplin 罐中, 60% 乙醇为1分钟。</li>
	</ol>
</li>
</ol>2. 制备 RNA 探针杂交的幻灯片前处理
<ol><li>在室温下进行以下洗涤, 除非另有规定。以低速速度在轨道振动筛上进行室温清洗。使用无菌幻灯片邮寄与房间多达五张幻灯片洗涤幻灯片。</li>
	<li>将幻灯片转移到无菌幻灯片邮件, 18 毫升的1x 磷酸盐缓冲盐水 (PBS)。室温下洗5分钟。</li>
	<li>倒入 1x PBS, 并立即用蛋白酶 K 处理幻灯片上的组织, 以使探针穿透组织。添加大约18毫升蛋白酶 K 溶液的幻灯片邮件 (见表 1的工作溶液浓度)。孵育37°c 15 分钟。不要动摇。<ol>
<li>当幻灯片正在孵化时, 准备 prehybridization 缓冲区 (Prehybe 缓冲区)。将 Prehybe 缓冲液放在沸水浴中10分钟, 然后在冰浴上冷却5分钟。有关 Prehybe 缓冲区的食谱, 请参见表 1。</li>
		<li>通过浇注蛋白酶 k 溶液停止消化蛋白酶 k 反应, 并立即将18毫升0.2% 甘氨酸 PBS 溶液添加到滑动邮件中。让幻灯片邮件在室温下坐5分钟。</li>
		<li>倒出0.2% 甘氨酸 PBS 解决方案, 并添加18毫升的2x 盐水柠檬酸钠 (SSC) 解决方案, 每个幻灯片邮件。在室温下将 2x SSC 中的幻灯片洗净10分钟, 以100-150 转每分钟的抖动为中心。</li>
	</ol>
</li>
</ol>3. Prehybridization 在 RNA 探针杂交制备中的研究进展
<ol><li>当幻灯片使用 2x SSC 进行清洗时, 获取新的无菌幻灯片邮件, 并将大约18毫升的 Prehybe 缓冲区放入幻灯片邮件中。在杂交温度下将滑动邮件放入杂交烤箱。 注: 杂交温度将取决于所使用的探针, 但通常范围从50-60 °c。</li>
	<li>一旦 2x ssc 清洗完成, 倒出 2x SSC, 并把幻灯片在 Prehybe 缓冲区中的幻灯片邮件在杂交烤箱1小时。</li>
</ol>4. RNA 探针的杂交
<ol><li>在杂交烤箱中用 Prehybe 缓冲器孵化时, 准备杂交探针溶液。<ol>
<li>稀释每个探针在杂交缓冲 (0.5 µL 探针与24.5 µL 的杂交缓冲)。 注: 杂交缓冲配方在表 1中找到。在特雷勒中可以找到探针的制备和浓度的例子. 11。</li>
		<li>将稀释探针放在86-90 摄氏度的散热块上12分钟, 然后在冰上冷却1分钟。</li>
	</ol>
</li>
	<li>从杂交烤箱中取出幻灯片邮件, 然后使用无菌镊子从幻灯片邮件中单独删除幻灯片。将幻灯片平放在纸巾上, 仔细擦拭组织样本周围多余的 Prehybe 缓冲。小心不要接触样品, 并迅速工作, 以防止干燥的组织样本。</li>
	<li>用 PAP 笔包围组织。应用25µL 稀释探针溶液与吸管和覆盖样品与塑料盖玻片。<ol>
<li>将样品放在滑水室中, 用 4x SSC + 50% 甲酰胺溶液在会议厅底部放置。</li>
		<li>一旦探头被添加到所有的幻灯片, 将滑动水分室放入杂交烤箱至少24小时在杂交温度为50-60 摄氏度。潜伏期可以根据探针的浓度而变化, 但至少要24小时。</li>
	</ol>
</li>
	<li>在用稀释后的探针过夜孵化后, 从杂交烤箱中取出滑水室。<ol>
<li>从每张幻灯片中取出盖玻片, 小心不要取代组织。在1000µL 吸管, 添加1000µL 的 2x SSC 解决方案, 并轻轻地清洗滑动。</li>
		<li>将幻灯片放在无菌幻灯片邮件中, 18 毫升 2x SSC 解决方案, 室温5分钟。倒出解决方案, 并用18毫升新鲜的 2x SSC 取代它。在室温下再重复孵化5分钟, 轻轻摇动。</li>
		<li>倒出 2x SSC 解决方案。添加18毫升的 1x SSC 溶液, 并在室温下清洗5分钟。倒出 1x ssc 解决方案, 并用18毫升的新鲜 1x ssc 替换它。在室温下, 重复5分钟的孵化, 轻轻摇动。</li>
		<li>倒出 1x SSC。添加18毫升的 0.5x SSC 和洗涤10分钟, 在42摄氏度不摇晃。倒入 0.5x ssc, 并用18毫升的新鲜 0.5x ssc 替换它。再洗10分钟, 在42摄氏度不摇晃。</li>
	</ol>
</li>
</ol>5. RNA 探针的可视化
注: 对于可视化探头, BM 紫色将在开发过程中使用。然而, 在这一步之前, 需要几个洗涤来准备样品染色。
<ol><li>用18毫升碱性磷酸酶缓冲液 (AP 缓冲器) 冲洗幻灯片, 不氯化镁2 1 分钟. 倒出 AP 缓冲器, 不氯化镁2, 并添加18毫升1x 勃林格-曼海姆堵塞缓冲剂稀释与马来酸缓冲。在一张滑动的邮件中, 以柔和的震动, 在室温下孵化至少1小时。 注: 勃林格-曼海姆阻塞缓冲液和马来酸缓冲器是预制和购买在挖洗和块缓冲集 (可利用商业)。<ol>
<li>另外, 可以在4摄氏度的一夜之间进行孵化。较长的阻塞期将减少非特定绑定的外观。</li>
	</ol>
</li>
	<li>准备20毫升稀释挖抗辛-AP 的碎片 (抗挖抗体 = 2 µL 抗挖抗体 + 20 毫升1x 勃林格-曼海姆阻断缓冲)。这足够在1塑料幻灯片邮件中使用。如果要使用多个幻灯片邮件, 则必须准备更多此解决方案。</li>
	<li>将抗挖抗体添加到新的无菌幻灯片邮件, 并将幻灯片传送到带有反挖抗体解决方案的幻灯片邮件。在室温下孵育3小时, 轻轻摇动。</li>
	<li>孵化后, 倒出抗挖抗体, 用18毫升的 AP 缓冲器清洗幻灯片邮件中的幻灯片, 而不氯化镁2 5 分钟的柔和震动。</li>
	<li>在没有氯化镁2的情况下,倒出 ap 缓冲器, 并添加18毫升的 ap 缓冲器。用柔和的震动洗5分钟。5分钟孵化后, 将 ap 缓冲液倒掉, 用18毫升新鲜的 ap 缓冲液代替, 用柔和的震动冲洗5分钟。</li>
	<li>在黑暗中, 倒出 AP 缓冲区, 并添加18毫升的 BM 紫色的幻灯片邮件。在室温下在黑暗中孵化幻灯片, 检查紫色的颜色发展每½ h. 可视化的反应时间将根据正在开发的探针而变化。 注意: 用 5-溴-4-氯-3-哚磷酸 (BCIP) 和硝基蓝四氮唑 (NBT) 来代替 BM 紫可视化开发解决方案。有关说明, 请参见表 1 。</li>
	<li>通过将幻灯片转移到新的无菌滑动邮件收发器, 在室温下, 在黑暗中, 以18毫升的三 EDTA (TE) 缓冲器为5分钟, 停止颜色开发。</li>
	<li>倒出 TE 缓冲器, 并添加18毫升的 RNase 免费水的幻灯片邮件和洗涤1分钟, 在黑暗中。</li>
	<li>将幻灯片从水中取出, 在组织的边缘周围晾干。加入甘油安装培养基, 放置盖玻片。将幻灯片存储在4摄氏度, 直到图片准备就绪。</li>
</ol>

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作者没有什么可透露的。

本协议中描述的方法已从以前在医学和进化发展研究8、9、10、12、17中的工作中进行了修改。该协议的重点是在原位杂交的细微差别与挖掘标记 RNA 反意识探针的成年珊瑚, 已保存和嵌入石蜡。这种方法可以很容易地转移到生物样本以外的珊瑚, 也已石蜡嵌入。最后, 这?...

珊瑚是重要的生态系统建设者和重要的生物多样性的海洋和人类健康1,2,3。由于气候变化和其他人为的压力, 它们受到威胁, 许多珊瑚物种被认为是严重濒危的。因此, 需要细胞和分子工具来诊断珊瑚在压力下。此外, 对于在成人珊瑚组织中表达基因的地方, 几乎没有什么了解, 因此对这些基因的功能了解甚少。为了解决这个问题, 我们已经修改了原位杂交协议, 通常用于人类医学和进化发育生物学, 用于在成人珊瑚的石蜡嵌入组织样本。这项技术是最强大的, 当用于成年珊瑚已经经历了紧张的事件, 如接触热应力。然而, 这种技术可用于珊瑚的广泛的组织和生命阶段, 并不仅限于热应激珊瑚4,6,7。此外, 这种技术可以用于任何后生的组织或细胞只要有 cDNA 序列信息可用。
这种方法的目的是在成年珊瑚组织中可视化 RNA 探针, 这些细胞已经保存并嵌入石蜡切片并切片到幻灯片上。这种方法是一个强大的诊断工具, 允许在成人珊瑚组织中的核酸可视化。最初这种方法是为医疗诊断而开发的, 此后它已成为发展生物学和进化发育生物学8,9,10等领域的流行工具。当基因组和 transcriptomic 序列数据可用, 但空间基因表达模式未知时, 它也是一种关键方法, 特别是在非模型系统中。对于非模型系统的诊断工作, 这种技术是强大的, 因为它可以表明哪些细胞和组织表达感兴趣的基因, 并可以导致更有针对性的治疗方法8,9,10, 11,12。最后, 该技术在与定量基因表达数据配对时, 质量和功能更强11。
本文概述的方法将感兴趣的研究人员已经设计了辛 (挖) 标记的 RNA 探针 (这两种感觉和反义探针), 现在准备进行原位杂交探针到一个样本。为了执行这种方法, 每一个正在测试的探针都需要两个连续切片的石蜡珊瑚组织。一节将用于感官探针, 另一个用于反义探针。感官探测器将是指示非特定绑定的控件。如果在感官探针中观察到染色, 则反义探针并不特定于感兴趣的 RNA。探针可以为任何表达的基因设计。在本议定书中, 以前发现在珊瑚热应激期间表达的几个例子: FBJ 小鼠骨肉瘤病毒癌基因同源 b (Fos b), 激活蛋白 (AP1), 肿瘤坏死因子受体 41 (TNFR 41)11。使用挖掘标记的 RNA 探针比使用放射性探针更可取, 因为它们的处理更安全10。此外, 这项技术是高度敏感的, 可以执行广泛的组织和胚胎以外的热应力成人珊瑚13,14,15,16。

<table><tbody><tr><td>Denhardt&#039;s solution</td><td>Affymetrix</td><td>70468 50 ML</td><td></td></tr><tr><td>Bioworld Alkaline phosphatase buffer</td><td>Fisher</td><td>50-198-724</td><td></td></tr><tr><td>Slide mailers</td><td>Fisher</td><td>12-587-17B</td><td></td></tr><tr><td>Bioworld Alkaline phosphatase buffer</td><td>Fisher</td><td>50-198-724</td><td></td></tr><tr><td>50 mL Falcon tubes</td><td>Fisher</td><td>14-959-49A</td><td></td></tr><tr><td>UltraPure Salmon Sperm DNA solution</td><td>Invitrogen</td><td>15632-011</td><td></td></tr><tr><td>PBS - Phosphate-Buffered Saline (10X) pH 7.4</td><td>Invitrogen</td><td>AM9625</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td><td>Invitrogen</td><td>10977-023</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water, 10 x 500 mL</td><td>Invitrogen</td><td>10977-023</td><td></td></tr><tr><td>UltraPure Salmon Sperm DNA solution</td><td>Invitrogen</td><td>15632-011</td><td></td></tr><tr><td>Slide white apex superior adhesive</td><td>Leica Biosystems</td><td>3800080</td><td></td></tr><tr><td>PBS solution, pH 7.4</td><td>Life Technologies</td><td>10010072</td><td></td></tr><tr><td>Proteinase K, Molecular Grade, 2 mL</td><td>New England Biolabs</td><td>P8107S</td><td></td></tr><tr><td>Super Pap Pen Liquid Blocker</td><td>Promega</td><td>22309</td><td></td></tr><tr><td>DIG Anti-Digoxigenin-AP Fab fragments</td><td>Roche</td><td>11093274910</td><td></td></tr><tr><td>BM Purple, 100 mL</td><td>Roche</td><td>11442074001</td><td></td></tr><tr><td>DIG Wash and Block Buffer Set</td><td>Roche</td><td>11585762001</td><td></td></tr><tr><td>NBT/BCIP</td><td>Roche</td><td>11681451001</td><td></td></tr><tr><td>Formaldehyde solution, 500 mL size</td><td>Sigma-Aldrich</td><td>252549-500ML</td><td></td></tr><tr><td>SSC Buffer 20X concentration</td><td>Sigma-Aldrich</td><td>S6639-1L</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma-Aldrich</td><td>320102-100ML</td><td></td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>47670-250ML-F</td><td></td></tr><tr><td>Triethanolamine</td><td>Sigma-Aldrich</td><td>90279-100ML</td><td></td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma-Aldrich</td><td>H3149-10KU</td><td></td></tr><tr><td>Xylenes, AR (ACS), For Histological Use</td><td>VWR</td><td>MK866806</td><td></td></tr><tr><td>Ethanol</td><td>VWR</td><td>EM-EX0276-4S</td><td></td></tr><tr><td>TE buffer</td><td>VWR</td><td>PAV6232</td><td></td></tr><tr><td>hybridization oven</td><td>VWR</td><td>97005-252, 97005-254</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>89032-088</td><td></td></tr></tbody></table>

in situ hybridization techniques for paraffin-embedded adult coral samples

珊瑚是重要的海洋无脊椎动物, 对整个海洋健康和人类健康至关重要。然而, 由于海洋温度上升和海洋酸化等人类影响, 珊瑚越来越受到威胁。为了应对这些挑战, 细胞和分子生物学的进步已经证明对诊断珊瑚的健康至关重要。修改人类医学常用的一些技术可以大大提高研究人员治疗和拯救珊瑚的能力。为了解决这一问题, 现已将一种主要用于人类医学和进化发育生物学的原位杂交协议用于在压力下的成年珊瑚中使用。
该方法的目的是可视化的 RNA 探针在成人珊瑚组织, 已嵌入石蜡和切片到玻璃幻灯片的空间表达。该方法的重点是去除样品的石蜡和补液, 对样品进行预处理, 以保证样品的渗透性, 预杂交孵化, rna 探针的杂交, rna 探针的可视化。这是一个强有力的方法, 当使用非模型有机体发现特定基因的表达, 并且协议可以很容易地适应其他非模型有机体。然而, 该方法是有限的, 因为它主要是定性的, 因为表达强度可能会因在可视化步骤中使用的时间量和探针的浓度而异。此外, 需要有耐心, 因为这个协议可能需要5天 (而且在许多情况下, 更长的时间) 取决于所使用的探头。最后, 非特异背景染色是常见的, 但这一限制可以克服。

完成该协议后, 将会对表达 RNA 探针的细胞和组织进行识别。该协议的代表性结果为 AP-1、FosB 和 TNFR41。这些结果, 以前发表的特雷勒-诺里斯等。11、在暴露于热应激的成人珊瑚上显示 RNA 探针的空间表达。图 1给出了两种不同染色类型的例子。图 1A是一种弥漫性组织染色的例子。染色是在整个组织中发现?...

Watch this Scientific Journal Video about In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples at JoVE.com

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

本议定书的目的是对已嵌入石蜡切片的成人珊瑚样品进行原位杂交, 并将其切片到玻璃滑梯上。这是一种定性的方法, 用于可视化的 RNA 反感探针在石蜡嵌入组织的空间表达。

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

in-situ-hybridization-techniques-for-paraffin-embedded-adult-coral

Research

JoVE Journal

Environment

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

7.0K 次观看.  University of Miami, Rosenstiel School of Marine and Atmospheric Sciences. 本议定书的目的是对已嵌入石蜡切片的成人珊瑚样品进行原位杂交, 并将其切片到玻璃滑梯上。这是一种定性的方法, 用于可视化的 RNA 反感探针在石蜡嵌入组织的空间表达。

视频: In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps&#160;and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter&#160;scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation,&#160;(2) superior optical sectioning ability,&#160;and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.

An integrated suite of imaging techniques has been applied to determine polyp morphology and tissue structure in the Caribbean corals Montastraeaannularis and M. faveolata. Fluorescence, serial block face,&#160;and two-photon confocal laser scanning microscopy have identified lobate structure, polyp walls, and estimated chromatophore and zooxanthellae densities and distributions.

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues ...

科研

环境科学

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

神经科学

增强癌症组织样本中致癌驱动因素的 FISH 分析

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

癌症研究

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and free-swimming medusae. As exemplified in established models such as Hydra and Nematostella, stem cells and/or proliferative cells contribute to the development and regeneration of cnidarian polyps. However, the underlying cellular mechanisms in most jellyfish, particularly at the medusa stage, are largely unclear, and, thus, developing a robust method for identifying specific cell types is critical. This paper describes a protocol for visualizing stem-like proliferating cells in the hydrozoan jellyfish Cladonema pacificum. Cladonema medusae possess branched tentacles that continuously grow and maintain regenerative capacity throughout their adult stage, providing a unique platform with which to study the cellular mechanisms orchestrated by proliferating and/or stem-like cells. Whole-mount fluorescent in situ hybridization (FISH) using a stem cell marker allows for the detection of stem-like cells, while pulse labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), an S phase marker, enables the identification of proliferating cells. Combining both FISH and EdU labeling, we can detect actively proliferating stem-like cells on fixed animals, and this technique can be broadly applied to other animals, including non-model jellyfish species.

Fluorescent In Situ Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish Cladonema pacificum

Here, we describe a protocol for visualizing stem-like proliferating cells in the jellyfish Cladonema. Whole-mount fluorescent in situ hybridization with a stem cell marker allows for the detection of stem-like cells, and 5-ethynyl-2&#39;-deoxyuridine labeling enables the identification of proliferating cells. Together, actively proliferating stem-like cells can be detected.&#160;

太平洋水母干细胞样细胞的荧光原位杂交和5-乙炔基-2'-脱氧尿苷标记

Cnidarians, including sea anemones, corals, and jellyfish, exhibit diverse morphology and lifestyles that are manifested in sessile polyps and ...

发育生物学

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

生物学

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

原位杂交技术在植物中的精确定位成绩单

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em在原位</em&gt;改编为海洋软骨鱼类杂交整装胚胎发育和细胞组织学

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

原位 冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

就地石蜡镶嵌成人珊瑚样品的杂交技术

摘要

探索更多视频

此视频中的章节

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

结合亲脂性染料，原位杂交，免疫组织化学和组织学

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

太平洋水母干细胞样细胞的荧光原位杂交和5-乙炔基-2'-脱氧尿苷标记

全山原位杂交E8.5，E11.5小鼠胚胎

原位杂交技术在植物中的精确定位成绩单

RNA

原位冷冻切片斑马鱼胚胎中的杂交结合免疫组织化学

就地石蜡镶嵌成人珊瑚样品的杂交技术

摘要

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此视频中的章节

多模态光学显微镜方法在加勒比海礁珊瑚显示息肉组织形态和结构

结合亲脂性染料，原位杂交，免疫组织化学和组织学

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

太平洋水母干细胞样细胞的荧光原位杂交和5-乙炔基-2'-脱氧尿苷标记

全山原位杂交E8.5，E11.5小鼠胚胎

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RNA

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