取自发育研究杂交瘤细胞NICHD的和维护由美国爱荷华大学生物科学系的主持下开发银行（J.多德，TM Jessell和A.川）开发的单克隆抗体（15.3B9，PAX7） ，爱荷华州。 DP是从国家药物滥用研究所露丝Kirschstein奖1F32 DA021977 - 01A1收件人。这项工作是支持的玛格丽特M. Alkek基金会RHF。

一，原理概述： 本视频演示了在整个装载在鸡胚免疫组织化学的不同步骤。首先，胚胎是固定在煤灰[IHC1]。然后淬火，内源性过氧化物酶的活性[IHC2]。胚胎培养中的主要抗体[IHC3]。经过多次洗涤，胚胎是在二抗孵育[IHC4];显色反应显示使用民建联[IHC5并出现抗体染色橙[IHC6]。 <img src="/files/ftp_upload/old/956_2009_1_26_3/figure 1.png" alt="图1" width="600" height="573" />第1部分：固定的胚胎<ol><li>要执行整装免疫组织化学鸡胚，首先打开一个鸡蛋，攻壳钳和消除件的外壳。 </li><li>删除与钳厚白蛋白，粗钳倾斜卵黄囊使胚胎朝上。 </li><li>用细剪刀，切割围绕胚胎的卵黄囊平方米。从蛋黄用勺子取出胚胎，并在含有PBS菜的地方。 </li><li>一个夹层显微镜下，小心地取出膜，蛋黄和胚胎移植到一个新的菜含有PBS。 </li><li>钳和昆虫引脚引脚的胚胎，并吸出PBS的。替换，与4％的PBS PFA，并让胚胎在室温下修复的上半年。 </li></ol>第2部分：准备胚胎抗体的一步<ol><li>为了尽量减少潜在的微生物污染，使用以下步骤DDH 2 O 。 </li><li>胚胎固定后，吸出固定液，并妥善处置化学废物。填写菜用PBS。 </li><li>下，使用显微切割刀，切一个胚胎周围的广场删除胚外膜。取出钳虫引脚。 </li><li>引脚已被删除后，使用钝年底巴斯德吸管胚胎转移到闪烁瓶中含有与PBT（PBS pH值7.4，0.5％TRITON X）。 </li><li>洗净与PBT的胚胎，3次，每次10分钟。 </li><li>从闪烁瓶中取出PBT和替换PBTx含有0.3％H 2 O 2，以灭活内源性过氧化物酶的潜在。 </li><li>孵育在室温下2小时就nutator。 </li><li> PBT，3次，每次10分钟，然后3次，每次30分钟洗净的胚胎。 </li></ol>第3部分：抗体孵育<ol><li>与抗体染色胚胎，阻止缓冲区或1％BSA / 1％农工商/ PBT（我们使用，因为二级抗体山羊提出的农工商超市）的胚胎洗一小时开始。一小时在室温下孵育nutator。 </li><li>稀释阻塞的缓冲区，并在这2天在4 °上nutator彗星的解决方案培育胚胎的主要抗体1:1。小学抗体稀释倍数取决于初级抗体的选择。在这里，我们使用的发育研究杂交瘤细胞上清提供的银行，这是一种抗体。我们在封闭液稀释为1:1。 </li><li>接下来，执行3 PBT 10分钟洗涤，其次是在PBT洗涤3 1小时。 </li><li>洗涤后，在封闭液稀释的二级抗体1:2500（过氧化物酶标记的羊抗鼠IgG（H + L），孵化的胚胎，并在此解决方案在这种情况下，O / N在4 °彗星上nutator。 </li><li>执行3 10分钟的洗涤，在PBT 3 1小时在PBT洗涤。 </li></ol>第3部分：显色反应<ol><li>要发展的显色反应的染色胚胎，第一次执行2 Tris缓冲液（100MM三盐酸，pH值7.4）20分钟的清洗。 </li><li>同时民建联基板（3,3&#39; - 二氨基tetrahydrochloride）溶解在Tris缓冲500μg/ml通风柜下，保持在黑暗的解决方案，在冰上。 </li><li>小瓶含胚胎取出最后的Tris缓冲液洗和步骤3.25毫升DAB溶液取代。在20分钟就nutator保持黑暗。处理的Eppendorf提示中含有10％的漂白粉溶液，以净化民建联桶。 </li><li>同时准备了0.3％的股票卫生署2 H 2 O 2在冰上。 </li><li>加入50μL股票H 2 O 2小瓶民建联胚胎。请在黑暗。 1-2分钟后，显示器在显微镜下的反应。 </li></ol>第4部分：摄影和组织学胚胎处理<ol><li>当显色反应完成后，民建联在处置漂白水桶。更换5毫升自来水。 </li><li>进行10分钟的PBS洗。 </li><li>要处理摄影和蜡切片，脱水乙醇（25％，50％，75％和100％），每次10分钟。 </li><li>更换乙醇与0.5 - 1毫升雪松木油（这会令胚胎半透明）。雪松木油的摄影过程​​。 </li><li>摄影后，返回胚胎样品瓶和雪松木油取代100％的乙醇。重复乙醇一步。 </li><li>更换Ëthanol 5％快绿FCF 100％的乙醇，孵育3分钟（这一步将会使胚胎组织学可见）。替换快绿FCF解决方案100％的乙醇和组织学过程。 </li></ol>代表性的成果： <img src="/files/ftp_upload/old/956_2009_1_26_2/figure2.png" alt="图2" width="600" height="498" />在下面的例子所示，胚胎阶段的HH 10（A）12（B）和11（三）在解剖胚胎显示在新兴的神经crestas百富7的表达以及在体节和神经管（A，B）; （c）中，脊索的是标有15.3B9（不- 1）（发育研究杂交瘤细胞银行提供的抗体）。

<ol>
	<li>Yamada, T., Placzek, M., Tanaka, H., Dodd, J., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+pattern+in+the+developing+nervous+system:+Polarizing+activity+of+the+floor+plate+and+notochord.">Control of cell pattern in the developing nervous system: Polarizing activity of the floor plate and notochord.</a> Cell. 64, 635-647 (1991).</li><li>Streit, A., Stern, C. D., Thery, C., Ireland, G. W., Aparicio, S., Sharpe, M. J., Gherardi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+HGF/SF+in+neural+induction+and+its+expression+in+Hensen's+node+during+gastrulation.">A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation.</a> Development. 121, 813-824 (1995).</li><li>Basch, M., Bronner-Fraser, M., Garcia-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neural+crest+occurs+during+gastrulation+and+requires+Pax7.">Specification of neural crest occurs during gastrulation and requires Pax7.</a> Nature. 441, 218-222 (2006).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li></ol>

本视频演示了年轻的鸡胚在执行整个安装抗体染色的不同步骤。这个协议基本上是用于小鸡 2,3表征的新型抗体的空间和时间，以及已知的抗原标志物的使用，以确定胚胎畸形，侮辱4。

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Eggs</td>
<td>&nbsp;</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH4 (16 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
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<td>Marsh Automatic Incubator</td>
<td>Other</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
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<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
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<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>&nbsp;</td>
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<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
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<td> Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
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<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
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<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to cut embryo from surrounding membranes following fixation</td>
</tr>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
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<td>16% PFA</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
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<td>30% H2O2</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H1009</td>
<td>&nbsp;</td>
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<td>BSA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A3803</td>
<td>&nbsp;</td>
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<td>NGS</td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>005-000-121</td>
<td>&nbsp;</td>
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<td>Primary Antibodies</td>
<td>Reagent</td>
<td>Developmental studies Hybridoma Bank</td>
<td>4G11, 15.3B9, PAX7</td>
<td>For these antibodies, investgators used mouse donnors</td>
</tr>
<tr>
<td>Peroxidase conjugated-Goat Anti-Mouse IgG (H+L) </td>
<td>Reagent</td>
<td>Jackson Immuno</td>
<td>115-035-003</td>
<td>&nbsp;</td>
<tr>
<td>3,3&#x2019;-diaminobenzidine tetrahydrochloride</td>
<td>Reagent</td>
<td>Pierce</td>
<td>34001</td>
<td>Store at -20; Allow bottle to warm to RT before use.</td>
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<tr>
<td>Cedar wood oil</td>
<td>Reagent</td>
<td>Sigma</td>
<td>W522503</td>
<td>&nbsp;</td>
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<td>Fast green FCF</td>
<td>Reagent</td>
<td>Sigma</td>
<td>F7252</td>
<td>&nbsp;</td>
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<td>Minuten pins 0.2mm diam</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-20 </td>
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method for whole mount antibody staining in chick

鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，使其研究在发育中的大脑，神经管和体节抗体表达的理想工具。本视频演示了在整个安装使用HRP标记二抗抗体染色的不同步骤，首先，从鸡蛋解剖胚胎和多聚甲醛固定。二，内源性过氧化物酶的灭活胚胎，然后暴露的主要抗体。经过多次洗涤，胚胎培养，以辣根过氧化物酶标记的第二抗体。过氧化物酶活性，发现用二氨基联苯胺底物的反应。最后，胚胎是固定的，摄影和切片处理。在使用荧光抗体这种方法的优点是可以处理的胚胎蜡切片，从而使截面抗原的网站研究。这种方法最初是由简多德和汤姆Jessell<sup&gt; 1</sup&gt;。

Watch this Scientific Journal Video about Method for Whole Mount Antibody Staining in Chick at JoVE.com

Method for Whole Mount Antibody Staining in Chick

本视频演示了整个安装免疫组织化学，抗原的时空表达模式的方法是，可以在年轻的鸡胚可视。这种方法最初是由简多德和汤姆Jessell。

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

method-for-whole-mount-antibody-staining-in-chick

Research

JoVE Journal

Biology

15.7K 次观看.  Texas A&M University (TAMU). 本视频演示了整个安装免疫组织化学，抗原的时空表达模式的方法是，可以在年轻的鸡胚可视。这种方法最初是由简多德和汤姆Jessell。

视频: Method for Whole Mount Antibody Staining in Chick

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

双整装原位杂交技术在早期鸡胚

科研

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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 ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用"冻结开裂"

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a &#39;whole mount&#39; tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).

A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

The goal of this protocol is to provide users with a set of methods for the high-throughput decapsulation of Lymnaea stagnalis embryos and larvae in preparation for whole mount in situ hybridization, and for subsequent pre- and post-hybridization treatments.

整个山<em&gt;原位</em&gt;对腹足软体动物杂交方法<em&gt;静水椎实螺</em

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...